Methods and compositions for glyphosate tolerance in plants

Abstract

The present disclosure relates to novel methods and compositions for conferring glyphosate tolerance to plants. The disclosure also provides glyphosate tolerant plants, seeds, tissues, cells and plant parts comprising the modified EPSP synthase and a recombinant DNA molecule encoding the modified EPSP synthase, as well as methods of producing the glyphosate tolerant plants, seeds, tissues, cells and plant parts and uses thereof.

Description

Cross-reference to related applications

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 511,073, filed June 29, 2023, pursuant to 35 USC §119(e), which is incorporated herein by reference in its entirety. Technical Field

[0002] This disclosure relates to the field of biotechnology. More specifically, this disclosure relates to a recombinant DNA molecule encoding an engineered 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) that provides tolerance to the herbicide glyphosate. This disclosure also provides methods for engineering the enzyme to have improved herbicide tolerance and enzymatic efficiency.

[0003] Merging of sequence lists The sequence list contained in the document named MONS540WO_ST26.xml is 41.3 kilobytes (measured in MS-WINDOWS) and was created on May 22, 2024. It was submitted electronically and is incorporated herein by reference. Background Technology

[0004] Glyphosate, or N-phosphonomethylglycine, is a broad-spectrum foliar herbicide that inhibits 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase or EPSPS) in plants. EPSPS is part of the shikimate pathway in plants for the biosynthesis of folate and aromatic amino acids. EPSP synthases from different organisms have been classified into two classes based on glyphosate sensitivity. All plants possess glyphosate-sensitive class I EPSP synthases. Glyphosate-tolerant crops primarily utilize glyphosate-insensitive class II EPSPS from Agrobacterium sp. strain CP4. Glyphosate tolerance in crops allows for the use of glyphosate to control weeds while maintaining crop yield. The T102I-P106S double mutation in class I EPSPS from maize has been shown to confer insensitivity to glyphosate; however, these resistance-conferring mutations also reduce the catalytic efficiency of this essential enzyme, and thus lead to reduced plant growth in naturally expressed plants. Therefore, there is a need for novel plant EPSPS variants with improved glyphosate tolerance and high catalytic efficiency, as well as methods for their production and application. Summary of the Invention

[0005] This disclosure provides a recombinant DNA molecule encoding glyphosate-resistant 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS), wherein the EPSPS comprises at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, and T102I-P106S-P126S-M217L. -K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the amino acid substitutions are relative to the positions of the amino acid sequences provided as SEQ ID NO: 6. In some embodiments, the EPSPS is maize EPSPS. In some embodiments, the EPSPS confers increased glyphosate tolerance or increased enzymatic efficiency compared to EPSPS lacking the said combination. In other embodiments, the EPSPS comprises at least two of the said substitution combinations.

[0006] This disclosure also provides glyphosate-tolerant EPSPS comprising at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-P126S-M217L-K296R, T102I-P106S -V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the amino acid substitutions are relative to the positions of the amino acid sequences provided as SEQ ID NO: 6. In a particular embodiment, the EPSPS confers increased glyphosate tolerance or increased enzymatic efficiency compared to EPSPS lacking the aforementioned combination, or compared to EPSPS containing only T102I-P106S. In other embodiments, the EPSPS comprises at least two of the aforementioned substitution combinations.

[0007] This disclosure further provides a plant, seed, cell, plant part, or commercial product comprising a recombinant DNA molecule encoding glyphosate-resistant 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS), wherein the EPSPS comprises at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R ... S-P126S-M217L-K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the amino acid substitutions are relative to the positions of the amino acid sequences provided as SEQ ID NO: 6. In some embodiments, the plant exhibits increased glyphosate tolerance when compared to a plant lacking the aforementioned combinations. In some embodiments, the plant is a maize, soybean, cotton, canola, wheat, rice, alfalfa, sugar beet, rapeseed, or sugarcane plant.

[0008] This disclosure also provides a transgenic maize plant comprising a recombinant DNA molecule encoding glyphosate-tolerant 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS), wherein the EPSPS comprises at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, and T102I-P106S-P126S. S-M217L-K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the amino acid substitutions are relative to the positions of the amino acid sequences provided as SEQ ID NO: 6, and wherein EPSPS confers glyphosate tolerance to the transgenic maize plant. In an additional embodiment, this glyphosate tolerance is greater than the glyphosate tolerance conferred by EPSPS containing only T102I-P106S substitutions.

[0009] This disclosure also provides a seed, cell, or plant part of a transgenic maize plant comprising a recombinant DNA molecule encoding glyphosate-resistant 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS), wherein the EPSPS comprises at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R ... S-P126S-M217L-K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the amino acid substitutions are relative to the positions of the amino acid sequences provided as SEQ ID NO: 6, and wherein EPSPS confer glyphosate tolerance to transgenic maize plants, wherein the seeds, cells, or plant parts contain recombinant DNA molecules.

[0010] This disclosure further provides a method for conferring glyphosate tolerance to plants, comprising expressing glyphosate-tolerant EPSPS in plants, the EPSPS comprising at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-P126S-M217L-K 296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the amino acid substitutions are relative to the positions of the amino acid sequences provided as SEQ ID NO: 6. In a particular embodiment, the method includes introducing a recombinant DNA molecule encoding glyphosate-tolerant 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS) into the genome of a plant or its ancestor, wherein the EPSPS comprises at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S- P126S-M217L-K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the amino acid substitutions are relative to the positions of the amino acid sequences provided as SEQ ID NO: 6.

[0011] Furthermore, this disclosure provides a method for generating glyphosate-tolerant EPSPS, comprising introducing at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-K296R, T102I-P106S-P126S-M217L-K2 96R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the amino acid substitutions are located relative to the amino acid sequence provided as SEQ ID NO: 6. In some embodiments, the introduction is performed in vitro. In other embodiments, the introduction is performed in situ in a plant. In some embodiments, the introduction includes the use of at least a first-position-specific endonuclease. In additional embodiments, the method includes introducing at least two of the substitution combinations into EPSPS.

[0012] This disclosure also provides a method for controlling weeds in a plant growth zone, comprising contacting a plant growth zone comprising: a transgenic maize plant containing a recombinant DNA molecule encoding glyphosate-tolerant 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS), wherein the EPSPS comprises at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-P126S, T102I-P106S-K296R. 96R, T102I-P106S-P126S-M217L-K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the amino acid substitution positions are relative to SEQ ID NO: 6. The position of the amino acid sequence provided, wherein EPSPS confers glyphosate tolerance to the transgenic maize plant; or the seed of the transgenic maize plant containing a recombinant DNA molecule encoding glyphosate-tolerant 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS), wherein EPSPS contains at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R. T102I-P106S-P126S-M217L-K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the amino acid substitutions are relative to the positions of the amino acid sequences provided as SEQ ID NO: 6, and wherein EPSPS confer glyphosate tolerance to transgenic maize plants, wherein the seeds, cells, or plant parts contain recombinant DNA molecules, wherein the plants or seeds are tolerant to glyphosate, and wherein weeds are controlled in the plant growth area.

[0013] Additionally, this disclosure provides a yeast cell comprising the knockout of natural aro1 function and further comprising a recombinant DNA molecule containing a heterologous promoter operably linked to an aro1 coding sequence containing a D731A mutation, wherein the yeast cell cannot grow in a culture medium lacking aromatic amino acids. In some embodiments, the yeast cell further comprises a heterologous nucleic acid molecule encoding EPSPS, wherein the yeast cell is capable of growing in a culture medium lacking aromatic amino acids. In some embodiments, the EPSPS is glyphosate-tolerant corn EPSPS. In other embodiments, the yeast cell is *Saccharomyces cerevisiae* (Saccharomyces cerevisiae). Saccharomyces cerevisiae )cell.

[0014] This disclosure also provides a method for identifying glyphosate-tolerant EPSPS exhibiting improved growth, improved glyphosate tolerance, or improved enzymatic efficiency compared to wild-type EPSPS, comprising the steps of: a) obtaining a yeast cell containing a knockout of natural aro1 function and further containing a recombinant DNA molecule comprising a heterologous promoter operatively linked to an aro1 coding sequence containing a D731A mutation, wherein the yeast cell cannot grow in a culture medium free of aromatic amino acids; and b) identifying the yeast cell as capable of growing in the presence of glyphosate and thus containing glyphosate-tolerant EPSPS exhibiting improved growth, improved glyphosate tolerance, or improved enzymatic efficiency compared to wild-type EPSPS. In other embodiments, the method includes obtaining a population of yeast cells collectively containing a plurality of heterologous nucleic acid molecules encoding mutant EPSPS proteins, and identifying at least one of said yeast cells as exhibiting improved growth, improved glyphosate tolerance, or improved enzymatic efficiency. In some embodiments, the method further includes cloning nucleic acid molecules encoding glyphosate-tolerant EPSPS from yeast cells or their progeny.

[0015] This disclosure also provides a method for identifying a plant, seed, cell, or plant part as containing glyphosate tolerance, the method comprising applying glyphosate to the plant, seed, cell, or plant part containing a recombinant DNA molecule encoding glyphosate tolerance 5-enol-pyruvyl-shikimate 3-phosphate synthase (EPSPS), wherein the EPSPS contains at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I -P106S-K296R, T102I-P106S-P126S-M217L-K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the amino acid substitutions are relative to the positions of the amino acid sequences provided as SEQ ID NO: 6. In some embodiments, the method further includes applying glyphosate to a population of plants, seeds, cells, or plant parts. In other embodiments, the method includes applying glyphosate to cell cultures. Attached Figure Description

[0016] Figure 1 The growth of wild-type Saccharomyces cerevisiae BY4741 (Fig. A) and Saccharomyces cerevisiae strain sKR-024-Ptef-EPSPS (Fig. B) in 0 mM, 5 mM and 10 mM glyphosate, and the interaction between Saccharomyces cerevisiae and corn ( Zea mays Growth of EPSPS, maize EPSPS-P102S, maize EPSPS-T106I and maize EPSPS-T102I-P106S in 5 mM glyphosate (Fig. C).

[0017] Figure 2 Figure A: Schematic diagram of the generation of a four-functional ScARO1 by removing the EPSPS function. Glyphosate competitively inhibits EPSP synthase to prevent the production of aromatic amino acids (AAA). Figure B: Overview of wild-type five-functional ARO1 in yeast. The mutant D731A successfully eliminated the catalytic function of the EPSPS moiety while maintaining the integrity and function of the other domains.

[0018] Figure 3 Construction of Δaro1, aro1 D731A mutant and aro1 D731A mutant-ZmEPSPS strain, and growth of the resulting strains on solid media containing and without aromatic amino acids.

[0019] Figure 4 The development of a synthetic yeast model enables the growth dependence of heterologous EPSPS. Figure A: Schematic diagram of a synthetic yeast model with two plasmids. Figure B: Synthetic yeast hosts expressing ZmEPSPS (green curve) or lacking ZmEPSPS (gray curve). Data represent the average of three ternary replicates randomly selected from a single colony, and the shaded error bars represent SE.

[0020] Figure 5 : A schematic diagram of the flow cytometry analysis of Saccharomyces cerevisiae strain sKR-024 containing red fluorescent protein (RFP) and TIPS cultured in yeast basic medium containing 0, 2.5, 5, 7.5 or 10 mM glyphosate, and the resulting RFP fluorescence intensity.

[0021] Figure 6 : Adjusting the selection pressure on the synthetic yeast host through expression optimization. Figure A: Specific growth rates of EPSPS and TIPS when expressed in 0 mM glyphosate at different promoter strengths (promoter series). Figure B: Growth rates obtained in 5 mM glyphosate. The difference in growth rates between EPSPS and TIPS realizes the selection window. The Core1p promoter was chosen due to the more consistent growth characteristics and differences between wild-type and mutant EPSP synthases. Figure C: In the synthetic yeast model, the growth curves of the well-studied mutant P106S and TIPS are close to the maximum values ​​seen in plant tissues during field application compared to wild-type EPSPS variants grown in 0.5 mM glyphosate. TIPS showed the highest glyphosate tolerance. Figure D: Variants grown in 0 mM glyphosate in the synthetic yeast model. The growth defects caused by TIPS are evident. Absorbance at 600 nm was measured over time on a Tecan plate reader. Each column represents the average of 3 biological replicates, where the specific growth rate of each replicate was calculated individually. Error bars represent standard deviation.

[0022] Figure 7 The growth of novel glyphosate-resistant mutants starting from maize EPSPS and maize EPSPS TIPS, with individual selection of the maize EPSPS and maize EPSPS TIPS to observe independent outcomes toward improved glyphosate resistance and / or improved overall catalytic activity.

[0023] Figure 8 Growth of EPSPS low and medium mutation frequency mutagenesis libraries in 0 mM, 2.5 mM and 5 mM glyphosate.

[0024] Figure 9Growth of EPSPS TIPS low and medium mutation frequency mutagenesis libraries in 0 mM, 2.5 mM and 5 mM glyphosate.

[0025] Figure 10 Mutation outcomes from libraries with low and moderate mutation frequencies in EPSPS and TIPS backgrounds.

[0026] Figure 11 Mutation outcomes from libraries with low and moderate mutation frequencies in EPSPS and TIPS backgrounds.

[0027] Figure 12 Schematic diagram of a TIPS (med) culture not exposed to any strong selection, a TIPS (med) culture with a single exosome (outgrowth) in 2.5 mM glyphosate, and a TIPS (med) culture with multiple exosomes whose oscillation selection ended in 2.5 mM glyphosate.

[0028] Figure 13 Mutant outcome from TIPS (med) cultures that were not exposed to any strong selection.

[0029] Figure 14 Mutation outcome from TIPS(med) culture with a single exogenous organism in 2.5 mM glyphosate.

[0030] Figure 15 Mutation outcomes from TIPS (med) cultures where oscillatory selection terminated at 2.5 mM glyphosate.

[0031] Figure 16 The results of optimized bitrait selection under pressure, starting from the EPSPS background (Figure A) and TIPS background (Figure B). The highest hits from each selection stage are shown.

[0032] Figure 17 Comparison of exemplary variants produced by single-trait or bitrait selection pressure. Figure A: EPSPS evolutionary entry point mutation pool. Each point represents the mean growth rate of a triplicate biological replicate grown in 0 mM or 0.25 mM glyphosate (i.e., a level at which wild-type EPSPS cannot grow). Figure B: TIPS evolutionary entry point mutation pool. Each point represents the mean growth rate of a triplicate biological replicate grown in 0 mM or 5 mM glyphosate (i.e., a level that evaluates high glyphosate tolerance).

[0033] Figure 18 Growth of each combination of EPSPS, TIPS, and the mutant TIPS-P126S-M217L-K296R in 0 mM, 2.5 mM, and 5 mM glyphosate.

[0034] Figure 19 Growth of EPSPS, TIPS, TIPS-P126S, TIPS-K296R and TIPS-P126S-K296R in 0 mM, 2.5 mM and 5 mM glyphosate.

[0035] Figure 20 Growth of EPSPS, G101A, G101A-P126S, G101A-K296R and G101A-P126S-K296R in 0 mM and 5 mM glyphosate.

[0036] Figure 21 Growth of EPSPS, T102I, T102I-P126S and T102I-K296R in 0 mM and 5 mM glyphosate.

[0037] Figure 22 Growth of EPSPS, P106S, P106S-P126S and P106S-P126S-K296R in 0 mM and 5 mM glyphosate.

[0038] Figure 23 Specific growth rate of each EPSPS variant at increased glyphosate levels. Each heatmap square represents the average of 3 biological replicates.

[0039] Figure 24 : Glyphosate and S3P density in EPSPS X-ray crystallography.

[0040] Figure 25 The crystal structure of the mutated ZmEPSPS TIPS (PSKR) is highlighted. The original TIPS mutation is circled (yellow dashed line). Complete X-ray crystallographic data collection and refined statistics are provided in Table 3.

[0041] Figure 26 Location and identity of T102, P106, I102 and S106 residues in ZmEPSPS and the X-ray crystal structures of the indicated variants.

[0042] Figure 27 Location and identity of P126, K296, S126 and R296 residues in ZmEPSPS and the X-ray crystal structures of the indicated variants.

[0043] Figure 28Figure A) Wild-type maize plants were transformed using the indicated variants and TIPS as a positive control. Plants were regenerated plantlets on a medium containing glyphosate to select only those plants transformed with the indicated EPSPS variants and expressing glyphosate tolerance. After regeneration, plants were allowed to recover and screened for molecular weight and plant health. The resulting superior plants were transferred to a greenhouse for seed production, and the remaining plants were stimulated with glyphosate spray. Figure B) Only two EPSPS variants produced plants sufficient for glyphosate spray stimulation: the TIPS positive control and TIPS-P126S-K296R. Plants were stimulated with glyphosate spray, and damage was observed one week after treatment. Glyphosate spray damaged three out of ten TIPS control plants, while no visible damage was observed in the plants expressing TIPS-P126S-K296R. Figure C) TIPS plants show typical glyphosate damage due to incomplete tolerance, including plant dwarfing and leaf margin discoloration (red arrows), while TIPS-P126S-K296R plants do not show glyphosate damage.

[0044] Figure 29 Figure A) Greenhouse spray analysis results of non-GMO control in the next generation under a hybrid background 12 days after treatment. Figure B) Greenhouse spray analysis results of surviving engineered EPSPS TIPS P126S, M217L, and K296R variants in the next generation under a hybrid background 12 days after treatment. Figure C) Greenhouse spray analysis results of EPSPS TIPS P126S and K296R variants in the next generation under a hybrid background 12 days after treatment. Figure D) Greenhouse spray analysis results of TIPS positive control in the next generation under a hybrid background 12 days after treatment. Plants were treated with glyphosate (Roundup PowerMax® 3) during the V3 growth stage. Damage was assessed 18 days after treatment, and the figures are shown 12 days after treatment. Treatments were UTC (untreated control), 1x typical Roundup field application (1120 g / ha); and 2x typical Roundup field application (2240 ​​g / ha). Plants are displayed in pairs (two representative plants for each treatment), except for the UTC used for the experimental samples, which consist of only one plant.

[0045] Brief explanation of the sequence SEQ ID NO:1 is a TIPS nucleotide sequence including promoter, transport peptide, exon, intron and 3' UTR.

[0046] SEQ ID NO:2 is the nucleotide sequence of the maize EPSPS genome, which includes transport peptides, exons, and introns.

[0047] SEQ ID NO: 3 is the nucleotide sequence of the maize EPSPS genome that includes only the transport peptide and exons.

[0048] SEQ ID NO: 4 is the nucleotide sequence of the maize EPSPS genome containing only exons.

[0049] SEQ ID NO:5 is the amino acid sequence of corn EPSPS containing a transport peptide.

[0050] SEQ ID NO: 6 is the amino acid sequence of corn EPSPS without the transport peptide.

[0051] SEQ ID NO: 7 is the nucleotide sequence of the maize EPSPS T102I-P106S (TIPS) genome, which includes only exons.

[0052] SEQ ID NO:8 is the amino acid sequence of TIPS containing a transport peptide.

[0053] SEQ ID NO: 9 is the amino acid sequence of TIPS without the transport peptide.

[0054] SEQ ID NO:10 is the TIPS-P126S-M217L-K296R amino acid sequence without the transport peptide.

[0055] SEQ ID NO: 11 is the amino acid sequence TIPS-P126S-M217L-K296R without the transport peptide.

[0056] SEQ ID NO: 12 is the T102I-P126S-K296R amino acid sequence without the transport peptide.

[0057] SEQ ID NO: 13 is the P106S-P126S-K296R amino acid sequence without the transport peptide.

[0058] SEQ ID NO: 14 is the P126S-K296R amino acid sequence without the transport peptide.

[0059] SEQ ID NO: 15 is the TIPS-P126S amino acid sequence without the transport peptide.

[0060] SEQ ID NO: 16 is the TIPS-K296R amino acid sequence without the transport peptide.

[0061] SEQ ID NO: 17 is the amino acid sequence of TIPS-V66L-M104L without the transport peptide.

[0062] SEQ ID NO: 18 is the amino acid sequence of TIPS-T108S without the transport peptide.

[0063] SEQ ID NO: 19 is the amino acid sequence of TIPS-F150V-H318Y without the transport peptide.

[0064] SEQ ID NO: 20 is the TIPS-G116V amino acid sequence without the transport peptide.

[0065] SEQ ID NO: 21 is the amino acid sequence of TIPS-I177V without the transport peptide.

[0066] SEQ ID NO: 22 is the amino acid sequence of TIPS-P126L without the transport peptide.

[0067] SEQ ID NO: 23 is the P106S-A109V-I163K amino acid sequence without the transport peptide. Detailed Implementation

[0068] 5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the reaction of phosphoenolpyruvate (PEP) with shikimate-3-phosphate (S3P) to produce phosphate and 5-enolpyruvylshikimate-3-phosphate (EPSP). This enzyme has been extensively studied due to its central role in the biosynthesis of aromatic amino acids and its tendency to be inhibited by the common herbicide glyphosate. Glyphosate's effectiveness in inhibiting the binding of PEP to EPSPS across all plant species, its simple synthetic pathway, and its overall low cost have made it the most commonly used herbicide in history. Since the 1990s, the discovery of highly glyphosate-resistant bacterial EPSPS enzymes from Agrobacterium strain CP4 has enabled the design of transgenic crops. Subsequent detailed structural and biochemical studies have led to the discovery and rational design of other glyphosate-resistant mutants. Herbicide-tolerant transgenic crops have enabled better weed control and have therefore been widely adopted in many regions, including the United States, where 90% of soybean, corn, and cotton growth contains this trait.

[0069] To date, only one set of mutations is known to confer glyphosate resistance to EPSPS and related homologues in natural plants; these resistance-conferring mutations also reduce the net activity of this essential enzyme, thus leading to reduced plant growth in naturally expressed plants. Therefore, glyphosate insensitivity in class I EPSPS enzymes severely impairs catalytic efficiency.

[0070] This disclosure overcomes the limitations known in the art by providing the following: exhibiting glyphosate resistance and improved catalytic properties (e.g., k cat / K mNovel engineered EPSP synthases; recombinant DNA molecules encoding said EPSP synthases; and components and methods for using and producing said recombinant DNA molecules. Cells, plants, and seeds expressing the engineered EPSP synthases of this disclosure exhibit improved glyphosate tolerance and can be used in agricultural methods such as weed control and crop production.

[0071] The following definitions and methods are provided to better define this disclosure and to guide those skilled in the art in practicing it. Unless otherwise stated, the terminology should be understood according to its conventional usage by those skilled in the art.

[0072] This disclosure provides novel engineered proteins and recombinant DNA molecules encoding said proteins. As used herein, the term "engineered" refers to non-natural DNA, proteins, cells, or organisms that are not normally found in nature and are produced through human intervention. "Engineered protein," "engineered enzyme," or "engineered EPSPS" refers to a protein, enzyme, or EPSPS that produces an amino acid sequence using one or more of the following techniques in a laboratory setting: biotechnology, protein design, or protein engineering, such as molecular biology, protein biochemistry, bacterial transformation, plant transformation, site-directed mutagenesis, directed evolution using random mutagenesis, genome editing, gene cloning, DNA ligation, DNA synthesis, protein synthesis, and DNA shuffling. For example, an engineered protein may have one or more deletions, insertions, or substitutions relative to the wild-type amino acid sequence of the protein, and each deletion, insertion, or substitution may consist of one or more amino acids. For example, genetic engineering can be used to produce DNA molecules encoding engineered proteins, such as engineered EPSPS that are glyphosate-resistant and contain at least a first amino acid substitution relative to the wild-type EPSPS protein as described herein.

[0073] The example of an engineered protein provided in this article is maize EPSP synthase, which contains one or more amino acid substitutions selected from the following: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-P126S-M217L-K296R, T102I-P106S-V66. L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K and combinations thereof, wherein the position of the one or more amino acid substitutions is relative to the position of the amino acid sequence provided as SEQ ID NO: 6.

[0074] In certain embodiments, the engineered proteins provided herein comprise one, two, three, four, five, six, seven, eight, nine, ten or more of any combination of such substitutions. Examples of such combinations include, but are not limited to: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-P126S-M217L-K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K.

[0075] In one embodiment, the engineered protein provided by this disclosure is an EPSP synthase conferred with glyphosate tolerance. As used herein, “EPSPS” refers to 5-enolpyruvylshikimate-3-phosphate synthase. EPSPS catalyzes the transfer of the enolpyruvyl moiety of phosphoenolpyruvate (PEP) to the 5-hydroxyl group of 3-phosphate shikimate (S3P) to produce phosphate and 5-enolpyruvylshikimate-3-phosphate (EPSP). This reaction is part of the biosynthesis of aromatic amino acids via the shikimate pathway in bacteria, fungi, and plants. Glyphosate is a competitive inhibitor of PEP, which, upon binding to EPSPS, inhibits the catalysis that blocks the shikimate pathway.

[0076] Engineered proteins can be produced by altering or modifying wild-type proteins to generate new proteins with one or more modified features, such as novel combinations of useful protein features, like altered V. max k cat K m K i IC 50 Substrate specificity, inhibitor / herbicide specificity, substrate selectivity, ability to interact with other components in the cell (such as partner proteins or membranes), and protein stability, etc.

[0077] A change can be made at a specific amino acid position in a protein by replacing an amino acid found at that position in the wild-type protein sequence with a substitute amino acid. As used herein, the term “substitution” or “substituting” refers to replacing one amino acid with another. Substitutions are indicated in standard scientific nomenclature by X#Y (where X is the original or wild-type amino acid, # is the amino acid position in the protein's amino acid sequence, and Y is the amino acid to be substituted for X). The DNA sequence encoding EPSP synthase having one or more amino acid substitutions as described herein can be generated by introducing changes into the DNA sequence encoding EPSPS using methods known in the art and the information provided in Table 1.

[0078] Table 1 Standard bioinformatics and sequence analysis tools (such as Clustal software tools or implementations of the Needleman-Wunsch or Smith-Waterman algorithms) can be used to align and compare EPSPS sequences from different plant species with SEQ ID NO: 6. Therefore, one or more substitutions described herein can be performed in any plant EPSP synthase by aligning the amino acid sequence of the target plant EPSPS with SEQ ID NO: 6, identifying the equivalent position of the desired substituted amino acid in the target EPSPS sequence relative to the specific amino acid position shown in SEQ ID NO: 6, and performing the substitution using the methods described herein.

[0079] Amino acid changes can be performed as a single amino acid substitution in a protein, or in combination with one or more other changes or mutations (such as one or more other amino acid substitutions, deletions, or additions). Proteins can be altered or modified by changing the DNA sequence encoding the protein, through one or more substitutions of the amino acid sequence relative to a reference sequence (such as a wild-type sequence). The alteration or modification can be made by any method known to those skilled in the art. In one embodiment, this disclosure provides engineered proteins, such as EPSPS, having one or more amino acid substitutions selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-M217L-K296R, T102I-P106S- V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, and combinations thereof, wherein the position of the one or more amino acid substitutions is relative to the position of the amino acid sequence provided as SEQ ID NO: 6. In a particular embodiment, the engineered protein provided herein comprises one, two, three, four, five, six, seven, eight, nine, ten, or more of any combination of such substitutions.

[0080] In another embodiment, this disclosure provides an engineered protein comprising one or more of the amino acid substitutions described herein, and recombinant DNA encoding the protein, the protein having at least about 85% sequence identity, about 90% sequence identity, about 91% sequence identity, about 92% sequence identity, about 93% sequence identity, about 94% sequence identity, about 95% sequence identity, about 96% sequence identity, about 97% sequence identity, about 98% sequence identity, about 99% sequence identity, about 99.5% sequence identity, about 99.8% sequence identity, and about 99.9% sequence identity with SEQ ID NO: 6.

[0081] Therefore, in some embodiments, the engineered protein provided by this disclosure provides engineered EPSPS with one or more altered protein characteristics relative to similar EPSPS or wild-type EPSPS found in nature. In one embodiment of this disclosure, such altered protein characteristics may include those that result in reduced sensitivity to glyphosate or increased tolerance or improved enzyme kinetics compared to similar wild-type EPSPS (e.g., EPSPS containing the sequence of SEQ ID NO: 6).

[0082] Therefore, these EPSPS variants or engineered EPSP synthases exhibiting reduced affinity for glyphosate while maintaining catalytic efficiency offer a method for achieving glyphosate tolerance in crops. EPSPS variants or engineered EPSP synthases can be measured by measuring the enzyme's maximum rate (Vt). max (represents the rate at which an enzyme catalyzes a reaction under substrate saturation conditions), and the turn number (k). cat Michaelis-Menten constant (K) m (representing the substrate concentration at which the enzyme's catalytic capacity is half) and the second-order rate constant (k) cat / K m The high proportion of carbon flux via the shikimic acid pathway requires highly efficient EPSPS (maximum catalytic efficiency) to prevent metabolic limitations or bottlenecks required by a variety of growth conditions at various developmental stages.

[0083] As used herein, the term "recombinant" refers to non-naturally occurring DNA, proteins, cells, seeds, or organisms produced through human intervention as a result of genetic engineering or genome editing and therefore not typically found in nature. "Recombinant DNA molecule" is a DNA molecule containing a DNA sequence resulting from human intervention, such as an engineered DNA molecule or a DNA molecule encoding an engineered protein or an engineered enzyme. Another example is a DNA molecule containing a combination of at least two heterologous DNA molecules (such as a DNA molecule encoding a protein and an operatively linked heterologous promoter). Another example is a DNA molecule encoding an EPSPS protein containing one or more amino acid substitutions as described herein. "Recombinant protein" is a protein containing an amino acid sequence resulting from human intervention, such as an engineered protein. Recombinant cells, seeds, or organisms are cells, seeds, plants, or plant parts containing a modified genome, produced by the use of genome editing techniques or plant transformation techniques, such as plant cells, seeds, plants, or plant parts containing the DNA molecules or proteins of this disclosure.

[0084] As used herein, “wildtype” means a naturally occurring or commonly occurring form. A “wildtype DNA molecule” or “wildtype protein” is a type of DNA molecule or protein that is naturally occurring or commonly occurring. For crop plants, this will be the type of DNA molecule or protein commonly found in that crop. The DNA sequence or amino acid sequence of the wildtype DNA molecule or protein is a sequence commonly found in that crop. The wildtype form of a DNA molecule or protein can be used as a reference DNA molecule or reference protein for comparison with recombinant or engineered DNA molecules or proteins. An example of a wildtype protein that can be used for comparison with engineered proteins provided by this disclosure is EPSPS from maize, provided as SEQ ID NO: 6. Other wildtype EPSP synthases that can be used for comparison with engineered proteins provided by this disclosure are known from other plants.

[0085] “Wild-type plants” are naturally occurring plants. Such wild-type plants can also be used for comparison with plants containing recombinant or engineered DNA molecules or proteins. Examples of wild-type plants that can be used for comparison with plants containing recombinant or engineered DNA molecules or proteins can be plants of the same type as plants containing engineered DNA molecules or proteins (such as proteins that confer herbicide tolerance traits), and therefore genetically different from plants containing herbicide tolerance traits. Examples of wild-type plants that can be used for comparison with maize plants include glyphosate-sensitive LH244 maize (ATCC registration number PTA-1173, ATCC®, Manassas, Virginia USA).

[0086] In some implementations, wild-type plants, or referred to as “control plants,” may also be used. As used herein, “control” means an experimental control designed for comparative purposes. For example, a control plant is a plant of the same type as the experimental plant (i.e., the plant to be tested) but without any transgenic inserts, recombinant DNA molecules, or genomic modifications of the experimental plant.

[0087] As used herein, the term “DNA” or “DNA molecule” refers to a genomic or synthetically derived double-stranded DNA molecule (i.e., a polymer or polynucleotide molecule of deoxyribonucleotide bases) read from its 5' (upstream) end to its 3' (downstream) end. As used herein, the term “DNA sequence” refers to the nucleotide sequence of a DNA molecule. The nomenclature used herein corresponds to the nomenclature shown in Tables (Tables 1 and 3) of Section 1.822, Title 37 of the United States Code of Federal Regulations and Appendix 2 of WIPO Standard ST.25 (1998).

[0088] This disclosure provides a nucleic acid molecule encoding maize EPSPS having one or more amino acid substitutions selected from the following: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-P126S-M217L-K296R, T102I-P106S-V6 6L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V and P106S-A109V-I163K and combinations thereof, wherein the position of the one or more amino acid substitutions is relative to the position of the amino acid sequence provided as SEQ ID NO: 6.

[0089] As used herein, the term "DNA molecule encoding a protein" refers to a DNA molecule containing a DNA sequence that encodes a protein. A protein-coding DNA sequence (also called a "protein-coding sequence") consists of a series of trinucleotide sequences called codons, which serve as genetic information for the amino acid sequence that produces the protein. As used herein, the term "protein" refers to a chain of amino acids linked by peptide (amide) bonds and includes both polypeptide chains that are folded or aligned in a biologically functional manner and unfolded or unaligned polypeptide chains. As used herein, "sequence" means the sequential arrangement of nucleotides or amino acids. The boundaries of a protein-coding sequence are typically defined by a 5' translation start codon and a 3' translation stop codon.

[0090] As used herein, the term "isolated" means that a molecule is at least partially separated from other molecules with which it normally associates in its natural state. In one embodiment, the term "isolated" refers to a DNA molecule separated from the nucleic acids of a DNA molecule normally side-attached to a DNA molecule in its natural state. For example, a DNA molecule encoding a protein naturally found in bacteria is not present in the DNA of a bacterium with which the protein-encoding DNA molecule is naturally found. Thus, DNA molecules, for example, fused to or operatively linked to one or more other DNA molecules that would not associate with in nature due to recombinant DNA or plant transformation techniques, are considered isolated herein. Such molecules are considered isolated even when integrated into the chromosome of a host cell or present in a nucleic acid solution with other DNA molecules.

[0091] As disclosed herein, any number of methods well known to those skilled in the art can be used to isolate and manipulate DNA molecules or fragments thereof. For example, polymerase chain reaction (PCR) technology can be used to amplify a specific starting DNA molecule and / or produce variants of the original molecule. DNA molecules or fragments thereof can also be obtained by other techniques, such as the direct synthesis of fragments by chemical means, as is typically carried out using automated oligonucleotide synthesizers.

[0092] Due to the degeneracy of the genetic code, different DNA sequences can encode the same amino acid sequence. For example, Table 1 provides a universal genetic code map showing all possible mRNA triplet codons (where T in the DNA molecule is replaced by U in the RNA molecule) and the amino acid encoded by each codon. DNA sequences encoding EPSPS with the amino acid substitutions described herein can be generated by introducing variations or mutations into DNA sequences encoding wild-type EPSPS using methods known in the art and the information provided in Table 1. In view of this disclosure, generating alternative DNA sequences encoding engineered proteins as described herein is entirely within the capabilities of those skilled in the art. These variant or alternative DNA sequences are within the scope of the embodiments described herein. As used herein, references to "substantially identical" sequences refer to sequences encoding amino acid substitutions, deletions, additions, or insertions that do not substantially alter the functional activity of the protein encoded by the DNA molecule of the embodiments described herein. Allelic variants of nucleotide sequences encoding wild-type or engineered proteins are also covered within the scope of the embodiments described herein. Amino acid substitutions other than those explicitly exemplified or naturally occurring in wild-type or engineered EPSPS are also covered within the scope of the embodiments described herein, provided that the substituted EPSPS retain substantially the same functional activities described herein.

[0093] As used herein, the terms “sequence identity percentage” or “sequence identity %” refer to the percentage of identical nucleotides or amino acids in the linear polynucleotide or amino acid sequence of the reference (“query”) sequence (or its complementary strand) compared to the test (“subject”) sequence (or its complementary strand) when two sequences are optimally aligned (within the comparison window, the total number of appropriate nucleotide or amino acid insertions, deletions, or vacancies is less than 20% of the reference sequence). Optimal alignment of sequences for comparison windows is well known to those skilled in the art and can be performed using tools such as Smith and Waterman's local homology algorithm, Needleman and Wunsch's homology alignment algorithm, Pearson and Lipman's similarity search method, and computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA, which are available as GCG® Wisconsin Package® (Accelrys Inc., San Diego, CA), MEGAlign (DNAStar Inc., 1228 S. Park St., Madison, WI 53715), and MUSCLE (version 3.6) (RC Edgar, "MUSCLE: multiple sequence alignment with high accuracy and high throughput"). Nucleic Acids Research The sequence identity score is obtained from a portion of the sequence analysis software package (32(5):1792-7 (2004)), for example, using default parameters. The “identity score” of the aligned segment between the test sequence and the reference sequence is the number of consistent components shared by the two aligned sequences divided by the total number of components in the aligned reference sequence segment, i.e., the entire reference sequence or a smaller defined portion of the reference sequence. The sequence identity percentage is expressed as the identity score multiplied by 100. Comparisons of one or more sequences can be performed on the full-length sequence or a portion thereof, or on a longer sequence.

[0094] As used herein, a “DNA construct” is a recombinant DNA molecule containing two or more heterologous DNA sequences. DNA constructs can be used for transgenic expression and can be contained in vectors and plasmids. DNA constructs can be used in vectors for transformation, that is, the introduction of heterologous DNA into host cells to produce transgenic plants and cells, and can therefore also be contained in plasmid DNA or genomic DNA of transgenic plants, seeds, cells, or plant parts. As used herein, “vector” means any recombinant DNA molecule that can be used for bacterial or plant transformation purposes. The DNA molecules provided herein can, for example, be inserted into vectors as part of a construct having a DNA molecule operatively linked to a gene expression element that functions in plants to affect the expression of a protein encoded by that DNA molecule. Methods for constructing DNA constructs and vectors are well known in the art and described in detail in, for example, manuals and laboratory guides, including MR Green and J. Sambrook, “Molecular Cloning: A Laboratory Manual” (4th edition) ISBN: 978-1-936113-42-2, ColdSpring Harbor Laboratory Press, NY (2012). The components of the DNA construct or the vector containing the DNA construct include one or more gene expression elements operatively linked to a transcribed DNA sequence, such as: a promoter for expressing the operatively linked DNA, a DNA molecule encoding the operatively linked protein, and an operatively linked 3' untranslated region (UTR). Gene expression elements that can be used in practice with this disclosure include, but are not limited to, one or more of the following types of elements: promoter, 5' UTR, enhancer, leader sequence, cis-acting element, intron, targeting or transport sequence, 3' UTR, and one or more optional marker transgenes.

[0095] The term "transgenic" refers to a DNA molecule artificially incorporated into the genome of an organism due to human intervention, such as through plant transformation. As used herein, the term "transgenic" means containing a transgenic gene; for example, a "transgenic plant" refers to a plant whose genome contains a transgenic gene, and a "transgenic trait" refers to a characteristic or phenotype transmitted or conferred by the presence of a transgenic gene incorporated into the plant genome. Therefore, transgenic plants are distinctly different from their wild-type counterparts, and transgenic traits are traits not naturally found in wild-type plants. The transgenic plants of this disclosure comprise recombinant DNA molecules and engineered proteins provided by this disclosure.

[0096] As used herein, the term "heterologous" refers to a relationship between two or more things that are not normally related in nature, such as things derived from different sources or things that are not normally found together in nature in any other way. For example, a DNA molecule or protein may be heterologous relative to another DNA molecule, protein, cell, plant, seed, or organism if it is not normally found together in nature or in the same context. In some embodiments, the two DNA molecules are heterologous if a first DNA molecule and a second DNA molecule are not normally found together in nature in the same context. For example, a recombinant DNA molecule encoding a protein that is not normally found in nature, combined with an operably linked promoter, is heterologous relative to the operably linked promoter. Similarly, a protein that is not normally found in nature, combined with a second operably linked protein (such as a transport peptide), is heterologous relative to the second operably linked protein. In another embodiment, the recombinant DNA molecule encoding EPSPS is heterologous relative to the operatively linked promoter if the combination of such a molecule and the promoter is not normally found in nature. The recombinant DNA molecule may also be heterologous relative to the cell, seed, or organism into which it is inserted, if it is not naturally present.

[0097] "Heterologous protein" is a protein present in a plant, seed, cell, tissue, or organism that is not naturally occurring or operatively linked to a protein not naturally occurring thereto. An example of a heterologous protein is an engineered EPSPS protein containing at least the first amino acid substitution described herein, expressed in any plant, seed, cell, tissue, or organism. Another example is a protein operatively linked to a second protein not naturally occurring thereto (such as a transport peptide or herbicide-resistant protein), or a protein introduced into a plant cell using genetic engineering techniques (which is not naturally occurring in that plant cell).

[0098] As used herein, “operably linked” means that two or more DNA molecules or two or more proteins are linked in such a way that one can affect the function of the other. Operablely linked DNA molecules or operablely linked proteins may be part of a single adjacent molecule and may or may not be adjacent. For example, in a DNA construct, a promoter is operably linked to a DNA molecule encoding a protein, wherein the two DNA molecules are arranged such that the promoter can affect the expression of the transgene.

[0099] The DNA constructs of this disclosure may include a promoter operatively linked to a DNA molecule encoding a protein provided by this disclosure, wherein the promoter drives the expression of the recombinant protein molecule. Promoters that can be used in practicing this disclosure include those that function in cells to express operatively linked polynucleotides, such as bacterial or plant promoters. Plant promoters are diverse and well known in the art, and include, for example, inducible promoters, viral promoters, synthetic promoters, constitutive promoters, time-regulated promoters, spatially regulated promoters, and / or temporally regulated promoters.

[0100] In one embodiment of this disclosure, the DNA construct provided herein includes a DNA sequence encoding a targeting sequence operatively linked to a heterologous DNA sequence encoding maize EPSPS, whereby the targeting sequence promotes intracellular localization of the polypeptide molecule. Target sequences are referred to in the art as signal sequences, targeting peptides, localization sequences, and transport peptides. Examples of targeting sequences are chloroplast transport peptides (CTPs), mitochondrial targeting sequences (MTS), or dual chloroplast and mitochondrial targeting or transport peptides. By promoting intracellular protein localization, targeting sequences can increase the accumulation of recombinant proteins, protect proteins from proteolytic degradation, and / or enhance herbicide tolerance levels, thereby reducing damage levels in cells, seeds, or organisms following herbicide application. CTPs and other targeting molecules that can be used in conjunction with this disclosure are well known in the art.

[0101] As used herein, “expression,” “expressing,” “protein expression,” and “expressing protein” refer to the production of a protein through the process of transcribing a DNA molecule into messenger RNA (mRNA) and translating the mRNA into a polypeptide chain, which ultimately folds into a protein. The DNA molecule encoding the protein is operatively linked to a heterologous promoter in a DNA construct for the expression of the protein in cells transformed with recombinant DNA molecules.

[0102] In one aspect, this disclosure provides cells, tissues, plants, and seeds comprising the recombinant DNA molecules of this disclosure or expressing engineered proteins of this disclosure, such as engineered EPSP synthase. These cells, tissues, plants, and seeds comprising the recombinant DNA molecules or engineered proteins exhibit tolerance to glyphosate.

[0103] One method for producing such cells, tissues, plants, and seeds is via plant transformation. Suitable methods for transforming host plant cells for use with this disclosure include any methods that introduce DNA into the cells (e.g., in which a recombinant DNA construct is stably integrated into a plant chromosome) and are well known in the art. Two effective and widely used cell transformation methods are Agrobacterium-mediated transformation and particle bombardment-mediated transformation. The particle bombardment method is illustrated in, for example, U.S. Patent Nos. 5,550,318; 5,538,880; 6,160,208; and 6,399,861. The Agrobacterium-mediated transformation method is described, for example, in U.S. Patent No. 5,591,616, which is incorporated herein by reference in its entirety.

[0104] Another approach to producing such cells, tissues, plants, and seeds is via genome editing. As used herein, the term “genome editing” refers to the modification of nucleotide sequences using genome editing methods and site-specific genome-modifying enzymes. Suitable methods for altering wild-type DNA sequences at predetermined chromosomal sites include any methods known in the art. Exemplary methods include the use of sequence-specific nucleases, such as zinc finger nucleases, engineered or naturally occurring meganucleases, TALE endonucleases, or RNA-guided endonucleases (e.g., the Clustered Regularly Interspersed Short Palindromic Repeat (CRISPR) / Cas9 system, CRISPR / Cpf1 system, CRISPR / CasX system, CRISPR / CasY system, or CRISPR / cascade system). Several embodiments involve genome editing methods that introduce precise base-pair modifications into the plant genome using single-stranded oligonucleotides, as described by Sauer et al. Plant Physiology As described in 170(4):1917-1928 (2016). Methods for genome editing, such as modifying, deleting, or inserting nucleic acid sequences into genomic DNA, are known in the art.

[0105] As used herein, in the context of plants, plant seeds, plant parts, plant cells, and / or plant genomes, "modified" refers to an engineered change in the expression level and / or endogenous sequence of one or more genes of interest relative to wild-type or control plants, plant seeds, plant parts, plant cells, and / or plant genomes. In practice, the term "modified" can further refer to plants, plant seeds, plant parts, plant cells, and / or plant genomes having one or more amino acid substitutions that affect the expression level and / or endogenous sequence of one or more genes of interest introduced via chemical mutagenesis, transposon insertion or excision, or any other known mutagenesis technique, or introduced via genome editing. EPSPS Genes. In one aspect, modified plants, plant seeds, plant parts, plant cells, and / or plant genomes may contain one or more transgenes. Therefore, for clarity, modified plants, plant seeds, plant parts, plant cells, and / or plant genomes include mutated, edited, and / or transgenic plants, plant seeds, plant parts, plant cells, and / or plant genomes that, relative to wild-type or control plants, plant seeds, plant parts, plant cells, and / or plant genomes, have… EPSPS Modified sequences of genes. Furthermore, in plants otherwise similar to those described above... EPSPS Compared to the activity of gene-encoded proteins, modifications can alter the activity of proteins encoded by genes. EPSPS The activity of genes-encoded proteins.

[0106] Modified plants, plant parts, seeds, etc., may have undergone mutagenesis, genome editing or site-directed integration, genetic transformation, or a combination thereof. Such "modified" plants, plant seeds, plant parts, and plant cells include those that are offspring or derived from "modified" plants, plant seeds, plant parts, and plant cells, and whose characteristics are preserved. EPSPS Molecular changes to genes (e.g., changes in expression levels and / or activity in the presence of glyphosate). The modified seeds provided herein can produce the modified plants provided herein. The modified plants, plant seeds, plant parts, plant cells, or plant genomes provided herein may contain recombinant DNA constructs or vectors or genome edits as provided herein.

[0107] Several embodiments involve plants containing modified EPSPS coding sequences in their genomes, wherein the modified EPSPS coding sequences encode glyphosate-resistant EPSPS as described herein. In some embodiments, genome editing methods are used to modify or replace existing coding sequences (such as EPSPS coding sequences) within the plant genome with sequences encoding engineered proteins (such as the engineered EPSPS coding sequences of this disclosure). In some embodiments, the natural EPSPS coding sequence is modified to include one or more targeted nucleotide changes, additions, deletions, or other modifications, such that the modified EPSPS coding sequence encodes glyphosate-resistant EPSPS comprising amino acid substitution combinations selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P10 ... 6S-M217L-K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K and combinations thereof, wherein the position of the one or more amino acid substitutions is relative to the position of the amino acid sequence provided as SEQ ID NO: 6.

[0108] Several implementations involve using known genome editing methods and site-specific genome-modifying enzymes, such as zinc finger nucleases, engineered or naturally occurring large-scale nucleases, TALE-endonucleases, or RNA-guided endonucleases (e.g., CRISPR / Cas9, CRISPR / Cpf1, CRISPR / CasX, CRISPR / CasY, CRISPR / cascade systems), to modify or replace existing EPSPS coding sequences in plant genomes. Therefore, several implementations involve providing site-specific genome-modifying enzymes capable of recognizing specific nucleotide sequences of interest within the plant genome, such as maize EPSPS sequences, to allow alteration of the EPSPS sequence through non-template-based or template-based editing.

[0109] Several implementation schemes involve recombinant DNA constructs containing expression cassettes encoding site-specific nucleases and / or any related proteins for genomic modification. These nuclease expression cassettes may coexist with a donor template for templated editing in the same molecule or vector, wherein the donor template is used to... Cis The method may be on a single molecule or a carrier ( transThe template DNA encodes glyphosate-resistant maize EPSPS proteins as described herein. Several methods for template-based editing are known in the art, involving different sequence-specific nucleases (or complexes of proteins and / or guide RNAs) that cleave genomic DNA to create double-strand breaks (DSBs) or nicks at desired genomic sites or loci. As understood in the art, during the process of repairing the DSBs or nicks introduced by the nucleases, donor template DNA can be integrated into the genome at the DSB or nick site.

[0110] As used herein, the term "site-specific genome-modifying enzyme" refers to any enzyme that can modify nucleotide sequences in a sequence-specific manner. In some embodiments, a site-specific genome-modifying enzyme modifies the genome by inducing single-strand breaks. In some embodiments, a site-specific genome-modifying enzyme modifies the genome by inducing double-strand breaks. In some embodiments, a site-specific genome-modifying enzyme comprises cytidine deaminase. In some embodiments, a site-specific genome-modifying enzyme comprises adenine deaminase. In this disclosure, site-specific genome-modifying enzymes include endonucleases, recombinases, transposases, deaminases, helicases, and any combination thereof. In some embodiments, a site-specific genome-modifying enzyme is a sequence-specific nuclease.

[0111] In one aspect, site-specific genome-modifying enzymes include endonucleases selected from: a wide range of nucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and Argonaute (non-restricted examples of Argonaute proteins include thermophilic bacteria). Thermus thermophilus Argonaute (TtAgo), Fierce Fireball ( Pyrococcus furiosus Argonaute (PfAgo), Halophilic bacillus griseus ( Natronobacterium gregoryiArgonaute (NgAgo)), RNA-guided nucleases, such as CRISPR-associated nucleases (non-restricted examples of CRISPR-associated nucleases include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12a) (also known as Cpf1), Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, CasX, CasY, their homologues or their modified forms).

[0112] In some embodiments, the site-specific genome-modifying enzyme includes a DNA-binding domain operatively linked to the deaminase. In some embodiments, the site-specific genome-modifying enzyme further includes a uracil DNA glycosidase (UGI). In some embodiments, the deaminase is a cytidine deaminase. In some embodiments, the deaminase is an adenine deaminase. In some embodiments, the deaminase is an APOBEC deaminase. In some embodiments, the deaminase is an activation-induced cytidine deaminase (AID). In some embodiments, the DNA-binding domain is a zinc finger DNA-binding domain, a TALE DNA-binding domain, a Cas9 nuclease, a Cas12a nuclease, a catalytically inactive Cas9 nuclease, a catalytically inactive Cas12a nuclease, a Cas9 nickase, or a Cpf1 nickase.

[0113] In some embodiments, the site-specific genome-modifying enzyme is a recombinase. Non-limiting examples of recombinases include tyrosine recombinases linked to the DNA recognition motifs provided herein, selected from the group consisting of Cre recombinase, Gin recombinase, Flp recombinase, and Tnp1 recombinase. In one aspect, the Cre or Gin recombinases provided herein are linked to a zinc finger DNA-binding domain, a TALE DNA-binding domain, or a Cas9 nuclease. In another aspect, serine recombinases linked to the DNA recognition motifs provided herein are selected from the group consisting of PhiC31 integrase, R4 integrase, and TP-901 integrase. In yet another aspect, DNA transposases linked to the DNA-binding domains provided herein are selected from the group consisting of TALE-piggyBac and TALE-mutant.

[0114] In one aspect, this disclosure provides glyphosate-resistant cells, plants, and seeds. Such cells, plants, and seeds can be used in agricultural methods such as weed control and crop production.

[0115] As used herein, “herbicide” is any molecule used to control, prevent, or interfere with the growth of one or more plants. Exemplary herbicides include 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitors (e.g., glyphosate), acetyl-CoA carboxylase (ACCase) inhibitors (e.g., aryloxyphenoxypropionate and cyclohexanedione), acetyllactone synthase (ALS) inhibitors (e.g., sulfonylureas, imidazolinones, triazolopyrimidines, and triazolinones), synthetic auxins (e.g., phenoxy compounds, benzoic acid, carboxylic acids, hemicarbazones), photosynthetic (photosystem II) inhibitors (e.g., triazines, triketones, nitriles, benzothiadiazoles, and ureas), glutamine synthase (GS) inhibitors (e.g., glufosinate and dipropionylphosphonate), and 4-hydroxyphenylpyruvate dioxygenase (…). HPPD inhibitors (e.g., isoxazole, pyrazolone, and trione), protoporphyrinogen oxidase (PPO) inhibitors (e.g., diphenyl ether, N-phenylphthalimide, aryltrione, and pyrimidinedione), very long-chain fatty acid inhibitors (e.g., chloroacetamide, oxyacetamide, and pyrazole), cellulose biosynthesis inhibitors (e.g., indaziflam), photosystem I inhibitors (e.g., paraquat), microtubule assembly inhibitors (e.g., pendimethalin), and phytoene desaturase (PDS) inhibitors (e.g., norflurazone).

[0116] As used herein, the term "glyphosate tolerance" or "glyphosate resistance" in relation to a protein means the ability to maintain at least some of its activity or function in the presence of glyphosate. For example, if EPSPS maintains at least some of its enzymatic activity in the presence of glyphosate, it is glyphosate resistant. Glyphosate tolerance can be measured by any means known in the art. For example, the enzymatic activity of EPSPS can be measured by bacterial analysis (such as the growth analysis described herein), in which recombinant EPSPS is expressed in bacterial cells that would otherwise lack EPSPS activity, and the ability of the recombinant EPSPS to supplement the knockout phenotype is measured. In another example, the enzymatic activity of EPSPS can be measured by analyzing enzyme kinetics in the presence and absence of glyphosate. Glyphosate tolerance can be complete or partial insensitivity to glyphosate.

[0117] As used herein, “glyphosate tolerance” or “glyphosate resistance” in relation to an organism, plant, seed, tissue, part, or cell means the ability of an organism, plant, seed, tissue, part, or cell to resist the toxic effects of glyphosate when applied. For example, glyphosate-tolerant plants can survive or continue to grow in the presence of glyphosate. Glyphosate tolerance in plants, seeds, plant tissues, plant parts, or cells can be measured by comparing the plant, seed, plant tissue, plant part, or cell to a suitable control. For example, glyphosate tolerance can be measured by applying glyphosate to a plant (test plant) containing a recombinant DNA molecule encoding modified EPSPS that confers glyphosate tolerance and to a plant (control plant) not containing a recombinant DNA molecule encoding modified EPSPS that confers glyphosate tolerance, and then comparing the damage rates of the two types of plants. The glyphosate tolerance of the test plant is indicated by the reduction in damage rate compared to the damage rate of the control plant. When compared with control plants, seeds, plant tissues, plant parts or cells, glyphosate-tolerant plants, seeds, plant tissues, plant parts or cells exhibit a reduced response to the toxic effects of glyphosate.

[0118] As used herein, "glyphosate tolerance trait" is a trait that confers improved glyphosate tolerance to a plant compared to a wild-type plant. Intended plants that can produce the glyphosate tolerance trait of this disclosure may include, for example, any plant, including monocotyledonous and dicotyledonous crops. Examples of monocotyledonous crops that can produce the glyphosate tolerance trait of this disclosure include, but are not limited to, maize and sorghum. Sorghum bicolor ),wheat( Triticum aestivum ),rye( Secale cereale ),banana( Musa paradisiaca L.), banana ( Musa sapientum L.), garlic ( Allium sativum ), like garlic ( Allium ampeloprasum ),onion( Allium cepa L.), rice ( Oryza sativa ),asparagus( Asparagus officinalis ),oat( Avena sativa L.) and barley ( Hordeum vulgare) Examples of dicotyledonous crop plants that can produce the glyphosate tolerance trait of this disclosure include, but are not limited to, those that can produce the glyphosate tolerance trait of this disclosure. Soybean (Glycine) Max, upland cotton (Gossypium hirsutum), sea island cotton (Goyssypium barbadense), rapeseed (Brassica napus) and Turnip (Brassica rapa).

[0119] As mentioned in this article, maize plants refer to those selected from the genus *Zea* (maize). Zea Any plant, including but not limited to any plant selected from the maize species.

[0120] As used in this article, “weed” is any unwanted plant. A plant can be considered generally unsuitable for agricultural or horticultural purposes (e.g., species of the genus *Amaranth*). Amaranthus Weeds may be species that are not desirable in certain circumstances (e.g., a crop plant of one species in a field of different species, also known as a free-growing plant). Weeds are generally known in the field and vary depending on geography, season, growing environment, and time. Lists of weed species can be obtained from agricultural and scientific societies (such as the Weed Science Society of America and the Canadian Weed Science Society), government agencies (such as the United States Department of Agriculture and the Australia Department of the Environment and Energy), and industry and farmers' associations (such as the United States Soybean Board, the National Corn Growers Association, and the Canola Council of Canada).

[0121] The application rate of herbicides may be the recommended commercial rate (1X) or any fraction or multiple thereof, such as twice the recommended commercial rate (2X). In some embodiments, the herbicide application rate may be expressed as grams per hectare (g / h) or pounds per acre (lbs / acre), pound acid equivalent per acre (lb ae / acre), gram acid equivalent per hectare (g ae / ha), pounds of active ingredient per acre (lb ai / acre), or grams of active ingredient per hectare (g ai / ha), depending on the herbicide and formulation. The plant growing area may or may not contain weeds when the herbicide is applied. The herbicide-effective dose of glyphosate in the area used to control weeds should consist of a range of about 0.1X to about 3X of the labeled application rate over the growing season. One (1) acre is equivalent to 2.47105 hectares and one (1) pound is equivalent to 453.592 grams. Herbicide dosage can be converted between imperial and metric units as follows: (lb ai / ac) multiplied by 1.12 = (kg ai / ha) and (kg ai / ha) multiplied by 0.89 = (lb ai / ac).

[0122] Herbicide application may be sequential, or may be mixed with one, two, or a combination of several herbicides or any other compatible herbicide. Multiple applications of one herbicide, or a combination of two or more herbicides alone, may be used during the growing season in areas containing the plants of this disclosure to control broad-spectrum dicotyledonous weeds, monocotyledonous weeds, or both, for example, two applications (such as pre-planting and post-emergence, or pre-emergence and post-emergence) or three applications (such as pre-planting, pre-emergence and post-emergence, or pre-emergence and two post-emergence applications).

[0123] Therefore, this disclosure provides a method for selectively controlling field weeds containing crops, which involves planting crop weeds or plants in the field that are resistant to glyphosate due to transformation by a recombinant DNA molecule or active variant thereof encoding EPSPS disclosed herein, or due to modification to include site-specific EPSPS genes disclosed herein; and applying sufficient amounts of glyphosate to the field crops and weeds to control the weeds without significantly affecting the crops.

[0124] The plants, offspring, seeds, plant cells, and plant parts disclosed herein may also contain one or more additional traits. Additional traits can be introduced by hybridizing a plant containing the recombinant DNA molecule provided by this disclosure with another plant containing one or more additional traits. As used herein, “hybridization” means propagating two individual plants to produce offspring plants. Two plants can be hybridized to produce offspring containing the desired trait from each parent. As used herein, “offspring” means any generation of offspring of the parent plants, and the offspring contain the herbicide tolerance trait provided by this disclosure and inherited from at least one parent plant. One or more additional traits can also be introduced by any means known in the art. Such additional traits include, but are not limited to, increased insect resistance, increased water use efficiency, increased yield efficiency, increased drought resistance, increased seed quality, improved nutritional quality, hybrid seed production, and herbicide tolerance, wherein the trait is measured relative to a wild-type plant. Exemplary additional herbicide tolerance traits may include transgenic or non-transgenic tolerance to one or more herbicides such as: ACCase inhibitors (e.g., aryloxyphenoxypropionate and cyclohexanedione), ALS inhibitors (e.g., sulfonylureas, imidazolinones, triazolopyrimidines, and triazolinones), EPSPS inhibitors (e.g., glyphosate), synthetic auxins (e.g., phenoxy compounds, benzoic acid, carboxylic acids, hemicarbazones), photosynthetic inhibitors (e.g., triazines, triketones, nitriles, benzothiadiazoles, and ureas), glutamine synthesis inhibitors (e.g., glufosinate), HPPD inhibitors (e.g., isoxazoles, pyrazolones, and triketones), PPO inhibitors (e.g., diphenyl ethers, N-phenylphthalimides, aryltriones, and pyrimidinidine diketones), and long-chain fatty acid inhibitors (e.g., chloroacetamide, oxyacetamide, and pyrazoles), etc. Exemplary insect-resistant traits may include resistance to one or more insect members within one or more of the following orders: Lepidoptera, Coleoptera, Hemiptera, Thysanoptera, Diptera, Hymenoptera, and Orthoptera. In view of this disclosure, such additional traits are known to those skilled in the art; for example, the Animal and Plant Health Inspection Service (APHIS) of the U.S. Department of Agriculture provides a list of such transgenic traits.

[0125] Glyphosate-tolerant plants and progeny can be used with any breeding method generally known in the art. In plant lines containing two or more traits, the traits may be independently segregated, linked, or a combination of both in plant lines containing three or more traits. Backcrossing with parental plants and crosscrossing with non-transgenic plants are considered, as with asexual reproduction. Descriptions of breeding methods commonly used for different traits and crops are well known to those skilled in the art. Various analyses can be performed to confirm the presence of one or more transgenic or genomic modifications in a plant or seed. Such analyses include, for example, molecular biological analyses such as Southern and Northern blotting, PCR, and DNA sequencing; biochemical analyses such as detecting the presence of protein products, for example by immunological means (ELISA and Western blot) or by enzymatic function; plant part analyses such as leaf or root analyses; and analyses of the whole plant phenotype.

[0126] As a result of backcrossing, the desired trait is introduced into the plant genotype. The plant genotype in which the trait has been introduced can be called a backcrossed genotype, strain, inbred line, or hybrid. Similarly, a plant genotype lacking the desired trait can be called an unconverted genotype, strain, inbred line, or hybrid.

[0127] As used in this article, the term "comprising" means "including but not limited to".

[0128] Several implementations have been described in detail, and it will be apparent that modifications, variations, and equivalent implementations are possible. Furthermore, it should be understood that the examples in this disclosure are provided as non-limiting examples.

[0129] Example Example 1: Evolution of bitrait EPSP synthase variants using a synthetic yeast selection system Identification of natural and mutant EPSPS can be achieved using novel gene-editing technologies, such as CRISPR / Cas, TILLING, and others, without the need for traditional biotechnology-based transgenic methods. To date, only one set of mutations is known to confer glyphosate resistance to natural plant EPSPS and related homologues. However, these resistance-conferring mutations also reduce the net activity of this essential enzyme, thus leading to reduced plant growth in naturally expressed plants. Glyphosate insensitivity has been well-established to severely impair catalytic efficiency in plant-derived enzymes and similar homologues (referred to as class I EPSPS enzymes). Commonly evaluated mutations (such as G101A, T102I, and P106S) all result in varying degrees of improved glyphosate tolerance, but are commercially unfeasible as single mutants due to the impaired enzymatic efficiency of G101A and T102I or the insufficient glyphosate tolerance of P106S. The dual mutants T102I and P106S (denoted as TIPS) are among the first commercially viable plant-derived glyphosate-resistant EPSPS mutants discovered. TIPS is a class I enzyme that is largely insensitive to glyphosate while still maintaining a reasonable K ratio for PEP. m Value. Even so, the number of transformations (k) cat It was significantly reduced in TIPS and manifested as a plant growth defect.

[0130] In a recent report, a glyphosate-resistant form of corn EPSPS with kinetics similar to the CP4 gene of Agrobacterium species was evolved by discovering at least 17 stacked mutations at different locations on the protein, starting with the G101A mutant and progressing through multiple rounds of evolution. Creating such a large number of mutations in plants using gene editing techniques may be challenging. Mutations that improve the catalytic efficiency of TIPS with fewer residue changes could establish commercially viable glyphosate-resistant EPSPS variants.

[0131] To meet the need for identifying novel EPSPS variants, a faster screening system than in situ plant or in vitro experiments is required. However, in Escherichia coli (E. coli) E. coli In other microbial model systems, subtle growth defects caused by known mutants in plants cannot be distinguished. For example, the mutant G101A exhibits growth very similar to wild-type EPSPS in an E. coli model, even though this variant has only about 1% of the catalytic efficiency of the wild-type enzyme and shows growth defects in plants.

[0132] This embodiment discloses the use of the eukaryotic model yeast *Saccharomyces cerevisiae* as a host system more suitable for the evolution of EPSPS variants. Yeast has been used to study certain herbicide interactions, including herbicides targeting acetyl-CoA carboxylase and other metabolic enzymes. Furthermore, the high similarity in aromatic amino acid biosynthesis between yeast and plants can facilitate the generation of novel targets for glyphosate tolerance. To establish this system, synthetic yeast strains that grow dependent on heterologous expression of EPSPS were generated. Next, a directed evolution strategy was used to simultaneously select for glyphosate resistance and improved catalytic properties (i.e., kJ / k ... cat / K m Both. It has been confirmed that additional mutations in the TIPS mutant background can significantly improve catalytic efficiency (k). cat / K m This approach maintains high glyphosate insensitivity while simultaneously demonstrating the importance of synthetic selection strategies for enzyme evolutionary trajectories used for bitrait optimization.

[0133] Materials and methods strains and culture media Yeast plasmids were propagated in *E. coli* DH10B. *Saccharomyces cerevisiae* strain BY4741 was obtained from the European Archive for Functional Analysis (EUROSCARF, Y00000). Yeast genomic DNA was extracted using the Wizard Genomic DNA Purification Kit (Promega). Yeast cells were routinely grown in YPD, YSC, and YMM media. YMM medium, used for selecting and characterizing EPSPS mutants, contained 1X YNB (6.7 g / L Yeast Nitrogen Base (YNB, Difco)), 1X glucose (20 g / L), 1X histidine, and 1X methionine. Solid media contained 2.0% agar. Yeast and bacterial strains were stored at -80°C in 2 mL Fisherbrand vials containing 20% ​​glycerol.

[0134] Transformation E. coli transformation is typically performed via electroporation at 2.5 kV in 2 mm Cuvettes (Bioexpress) using BioRad Genepulser Xcell. Usually, 100 ng of cloning product from Gibson assembly or ligation is added to 50 µL of competent cells. The transformant is rapidly recovered in 500 µL of SOC medium. The recovered culture is then plated or streaked onto LB agar plates containing 100 µg / mL ampicillin and incubated overnight at 37°C. The following day, single colonies are picked and grown overnight in 4 mL of LB medium containing 100 µg / mL ampicillin at 30°C. The following day, plasmids are isolated (GeneJET Plasmid Miniprep Kit, Thermo Scientific) and confirmed by Sanger sequencing.

[0135] Yeast transformation was performed routinely using the Frozen EZ Yeast Transformation II Kit (Zymo Research) according to the manufacturer's instructions. Generally, 25 µL of chemically competent *Saccharomyces cerevisiae* strain BY4741 was transformed with 100-500 ng of plasmid DNA. Cells were incubated at 30°C for 1-2 hours, followed by plating at 30°C on selective media YSC-Ura, YSC-Leu, and YSC-Ura-Leu for 2 days. Single colonies were then randomly picked into 1-2 mL of appropriate selective medium and grown at 30°C with shaking at 225 RPM for 2 days.

[0136] Plasmid and strain construction Oligonucleotides were synthesized using Integrated DNA Technologies. PCR was performed using Q5 hot-start polymerase (NEB). PCR and other DNA products were cleaned using NucleoSpin Gel and a PCR cleaning kit (Macherey Nagel). Vectors were digested using various restriction enzymes (NEB) as indicated and prepared for ligation using antarctic phosphatase (NEB). Unless otherwise specified, low-copy CEN / ARS vector backbones from the modified Mumberg set (p415-TEF1-PRM9t and p416-PTEF1-CYC1t with strong TEF1 promoters) were used for all initial experiments (Crook et al.). Nucleic Acids Res. 39:e92 (2011); Mumberg et al., Gene156:119-122 (1995)). ScARO1 was amplified by PCR from the genomic DNA of *Saccharomyces cerevisiae* BY4741 and cloned into the plasmid p415-PTEF1-PRM9t using the Gibson method for overexpression in *Saccharomyces cerevisiae* (Gibson, 1995). Nat. Methods 6:343-345 (2009)).

[0137] ZmEPSPS was codon-optimized for Saccharomyces cerevisiae to synthesize gBlock (IDT), and initially cloned into plasmid p416-PTEF1-CYC1t. These plasmids were named p415-TEF-ARO1mut (Saccharomyces cerevisiae endogenous gene) and p416-TEF-EPSPS (maize homologue). The TEF1 promoter was chosen for these vectors because it has been reported as a strong promoter, exhibiting moderately more consistent activity than other extremely strong promoters under different carbon sources and glucose concentrations (Partow et al.). Yeast 27:955-964 (2010)). The promoter series promoters, for the short core promoters Core1p and Core4p, are synthesized via oligonucleotide synthesis (IDT), or for STE5p and CYC1p, they are amplified from the Saccharomyces cerevisiae genome (Redden and Alper, 2010). Nat. Commun. 6:7810 (2015)). The resulting plasmids were named p416-Core1p (or other promoter)-EPSPS (low copy, CEN / ARS) and p415-TEF-ARO1mut (low copy, CEN / ARS).

[0138] Mutagenesis methods and library construction for ARO1 and EPSPS mutants ScARO1 point mutants were generated using the Agilent QuikChange Multisite Directed Mutagenesis Kit. ZmEPSPS were amplified using Agilent Mutazyme II to introduce mutations into coding sequences (CDS) to generate mutagenesis pools. Two mutation frequencies were targeted; approximately 1–3 and 3–5 mutations per CDS length, respectively, representing low and medium frequencies. The resulting PCR product pools were cloned into p416-Core1p via Gibson assembly or ligation with T4 DNA ligase and transformed into *E. coli* DH10B, yielding approximately 8 × 10⁸ cells per library. 5 Up to 4×10 6Several variants were selected, depending on the mutagenesis pool used for transformation into yeast (Mumberg et al., see above; Gibson, see above). DNA was isolated from ten individual colonies and sequenced, and for each library, 1–3 mutations were achieved on average per CDS at low frequency and 2–5 mutations were achieved on average per CDS at medium frequency; limited wild-type template sequences were observed. The low-copy-number plasmid p416-Core1p was selected as the acceptor vector for this experiment to minimize the potential impact of plasmid copy number variations and to ensure maximum growth differentiation when comparing mutant EPSPS sequences (Karim et al., FEMS Yeast Research 13:107-116 (2013)). After isolating DNA from E. coli, a highly efficient lithium acetate protocol (Gietz and Schietz, 13:107-116 (2013)) was used. Nat. Protoc. 2:31-34 (2007) transformed the library into Saccharomyces cerevisiae strain sKR-024, producing approximately 3 × 10⁻⁶ cells / mL. 5 Up to 2×10 6 A library of variants, depending on cloning and conversion efficiency.

[0139] Flow cytometry Yeast cultures were started directly from random colonies or from frozen stock solutions and transferred in triplicate to appropriate selective media YSC-Ura, YSC-Leu, and YSC-Ura-Leu. Cultures were incubated at 30°C for 48 hours to saturation. The fully grown yeast cultures were then diluted back to OD 0.01 and incubated at 30°C in a 96-well shaker for 48 hours. Fluorescence was then analyzed for mKate2 RFP using a BD Accuri C6 flow cytometer (BD Biosciences) at 588 nm excitation and 633 nm emission. The yeast populations of interest were then gated using logarithmic plots of side-scattered (SSC) and forward-scattered (FSC) fluorescence based on relative size and complexity, and spectra depicting RFP fluorescence intensity versus cell count were generated. All flow cytometry data were analyzed using FlowJo software.

[0140] Directed Evolution Workflow Yeast library pools were started from frozen stock solution and transferred to yeast basal media with or without glyphosate, depending on the applied selection criteria. Generally, the libraries were diluted back to OD 0.01 and allowed to grow until saturation, typically within 48–96 hours, depending on the mutants present in each pool. After saturation, a portion of the pools was cryopreserved in 20% glycerol and then subcultured into the next set of media, with or without glyphosate. Mutants were then isolated by selecting random colonies on glyphosate-containing or glyphosate-free agar plates, as indicated in the workflow diagram. Unless otherwise instructed, final selection was performed on glyphosate-free agar plates. The EPSPS library ORFS was isolated from random colonies surviving the glyphosate selection on the plate using a modified colony PCR method with a boiling step containing NaOH and Triton X-100. PCR was confirmed by gel electrophoresis, the plates were cleaned, and then Sanger sequencing was performed. The sequenced PCR product was then amplified and transformed into a clean strain of Saccharomyces cerevisiae sKR-024 to confirm the phenotype with three biological replicates.

[0141] In vivo characterization and growth rate analysis of EPSPS variants As described above, the EPSPS variant was re-cloned into a clean p416-Core1p backbone and transformed into strain sKR-024. The colonies were then picked in triplicate into YSC-Ura-Leu medium and incubated at 30°C for 2 days. The cultures were then diluted back to OD 0.001 in yeast basal medium (YMM) containing varying amounts of glyphosate, as indicated. The cultures were then incubated at 30°C for 72–120 hours according to the selected variant's growth rate, and growth rate measurements were obtained using an Infinite M200 PRO microplate reader (Tecan, Männedorf, Switzerland).

[0142] Statistical analysis All statistical analyses were performed using GraphPad Prism's built-in functions. One-way ANOVA was used to determine statistical differences between group means. Tukey's Post-Hoc test was used to compare pairs of group means. Biological replicates and significance levels were highlighted where appropriate.

[0143] In vitro characterization of EPSPS mutants The amount of P that is coupled with MESG was quantified using the EnzChek phosphate assay kit from Invitrogen (first released in 1992). iThe activity was determined by the release of purine-nucleoside phosphorylase, which catalyzes the reaction. 95 µL of the master mixture was added to each well, and 5 µL of different substrate dilutions were introduced into the plate via a multichannel to initiate the reaction simultaneously under all conditions. Absorbance was measured at 360 nm every 10 seconds for 3 minutes to capture the initial reaction rate for each condition. Different levels of substrate were added to produce the resulting reaction rate profiles. Each condition was performed in triplicate. i As previously stated (Lu et al., Protein Engineering, Design and Selection The data were fitted to the Michaelis-Menten equations and validated for each variant using built-in functions of GraphPad Prism. (30:395-399 (2017))

[0144] Protein expression and purification For protein expression of EPSPS and its variants, *E. coli* BL21 (DE3) was used as the expression host. Electrocompetent cells were transformed with the corresponding construct plasmid pET28a(+) containing the selected EPSPS variants (wild-type, TIPS, TIPS P126S, TIPS K296R, TIPS P126S / K296R). Single colonies of *E. coli* BL21 (DE3) containing one of the constructed plasmids were inoculated into 2 mL of Luria Bertani broth (LB) containing 50 µg / mL kanamycin and grown overnight at 37°C / 225 rpm. The overnight culture (using 100 µl) was scaled up by a 2000-fold dilution in a 500 mL three-baffle shake flask and grown to a cell density of 0.8 (optical density [OD600]) at 37°C / 225 rpm. Protein expression was induced by adding 0.2 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and cells were cultured at 20 °C / 225 rpm for 24 h. The induced cell culture was harvested by centrifugation at 4,000 g and 4 °C for 20 min. The cell pellet was then resuspended in 25 mL of Dulbecco's Phosphate Buffered Saline (DPBS) (Thermo Fisher Scientific, Waltham, MA) pH 7.0 buffer containing 10 mM imidazole, 1 g / L lysozyme, and 5 µl Pierce™ universal nuclease (Thermo Fisher Scientific, Waltham, MA), and then mixed on a shaker at 4 °C for 30 min. Subsequently, cells were lysed by sonication, and the resulting cell lysate was centrifuged at 14,000 g and 4 °C for 20 min to obtain a supernatant containing soluble proteins. Purify the target protein from the supernatant using HisPur™ Ni-NTA resin (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions.

[0145] The eluent was dialyzed against the appropriate buffer with 3C protease added to the dialysis cartridge, followed by size exclusion rapid protein HPLC. All EPSP synthase variants were stored in 20 mM Tris (pH 7.5), 100 mM NaCl, and 10 mM β-mercaptoethanol. Protein concentration was then determined by measuring the absorbance of the mixture using the Coomassie Plus Bradford assay kit (ThermoFisher Scientific) and an Infinite M200 PRO microplate reader (Tecan, Männedorf, Switzerland). The presence and purity of the purified proteins were assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

[0146] Protein crystallization To identify crystallization conditions for maize EPSP synthase variants, 20 mg / ml of purified wild-type EPSP synthase sample was pre-incubated at 4°C for 3 h with 5 mM shikimate-3-phosphate and 5 mM glyphosate, followed by screening using a sparse matrix sieve with the Phoenix robotic system (Art Robinson). Initial crystal hits were identified by the rod-shaped crystals that appeared after incubating the sieve plate at 25°C for three days. Crystallization conditions for all EPSP synthase variants were optimized to 0.1 M MES, 25% PEG 8000, pH 6.5, set for sitting-drop vapor diffusion. All sitting drops were set by mixing 1.33 μl of reservoir solution with 0.67 μl of 20 mg / ml protein sample.

[0147] X-ray diffraction data acquisition, data processing, and model refinement After cryoprotection with 30% (v / v) glycerol, single EPSP synthase crystals were rapidly frozen in liquid nitrogen. X-ray diffraction data were collected at the 23-ID-B beamline in an Advanced Photon Source (Lemont, IL). X-ray diffraction patterns of all EPSP synthase crystals were processed to approximately 2 Å resolution using an HKL2000 (Otwinowski and Minor, Meth. Enzymol. 276:307-326 (1997)). The structure of wild-type EPSP synthase was resolved by molecular substitution using an EPSP synthase from *E. coli* as the initial search model (PDB code 1G6T). Coot (Emsley et al.) was used. Acta Crystallogr. D Biol. Crystallogr.66:486-501 (2010)) and Phenix (Liebschner, Acta Cryst. D 75:861-877 (2019)) The refinement package iteratively constructs and refines molecular substitution resolution schemes for wild-type EPSP synthase. Crystal structures of other variants are resolved and iteratively refined using molecular substitutions with the wild-type EPSP synthase structure. Procheck and MolProbity are used to evaluate the quality of the final determined EPSP synthase structures. Final diffraction data collection and structure determination statistics are shown in Table 2.

[0148] Table 2 The value of the corresponding parameter in the outermost shell within the parentheses.

[0149] ϒ CC 1 / 2 The Pearson correlation coefficient is the result of a randomized half of the data set. The two numbers represent the lowest and highest resolution shells, respectively.

[0150] ± R free R is calculated for approximately 10% of reflections that are randomly selected and omitted during refinement. work .

[0151] The ^MolProbity score is calculated by combining the clash score with rotational isomers and Lagrange percentages, and scaled proportionally based on X-ray resolution. For structures with comparable resolution, the 100th percentile is used to calculate the percentage, with the 0th percentile representing the best and the 0th percentile representing the worst.

[0152] result Production of EPSPS-dependent host-selective Saccharomyces cerevisiae Previous studies have primarily focused on *Escherichia coli* as a basis for selecting modified EPSP synthases. This example details the development of *Saccharomyces cerevisiae* as a model eukaryotic system for glyphosate resistance, thus requiring the cells to be synthetically adapted for EPSP expression and activity in an effort to establish a growth-based selection protocol. Glyphosate resistance in yeast has been previously reported. This finding was validated by the observation that wild-type *Saccharomyces cerevisiae* BY4741 was relatively insensitive to large amounts of glyphosate and exhibited slight growth inhibition at concentrations of 5 mM and 10 mM, thus indicating that the native EPSP synthase subunit of ScARO1 is not strongly inhibited by this compound. Figure 1 ).

[0153] To establish a selection scheme, it is necessary to remove the native function of EPSPS and supplement the mutant form. However, since yeast ARO1 is pentafunctional and the EPSPS function is located in the middle of the protein, eliminating the function using previously developed methods (such as truncation) is not feasible. Figure 2 Elimination of ARO1 EPSPS function was accomplished by testing a series of alanine mutations and screening on a medium lacking aromatic amino acids. To guide mutation selection, mutants derived from *Chaetomium thermophilum* were used. Chaetomium thermophilum A structurally similar five-functional AROM complex was used as a template to generate a homology model for yeast ARO1p. Figure 2 Using this model, structural data from similar EPSPS enzymes were evaluated to identify mutations that might disrupt EPSPS function while preserving the function of the other four enzymes. This analysis evaluated mutations at residues D731 and C853, as well as a triplet mutant consisting of residues K425, S426, and R430. The analysis showed that individual mutants disrupt so-called key residues in the reaction mechanism, while the triplet mutant disrupts the entire active site.

[0154] Each of the three identified aro1 mutants was expressed on plasmid (p415-PTEF-PRM9t) in the *Saccharomyces cerevisiae* strain Δaro1::KanMX. The resulting strains were then plated on solid media containing and without aromatic amino acids to test growth complementation. Neither D731A nor the triple alanine substitution mutant grew on media lacking aromatic amino acids. Figure 3 C853A, while retaining its natural activity, was not a viable option. After identifying the putative invalid EPSPS allele, the same strain was transformed with a plasmid containing wild-type EPSPS (ZmEPSPS) from maize to observe whether aromatic amino acid synthesis could be restored through complementation. Expression of aro1-D731A effectively eliminated ScEPSPS function only, and growth could be reconstituted by supplementing ZmEPSPS. The final strain, containing the knockout of natural aro1 function and replaced with p415-PTEF-aro1(D731A)-PRM9t, was grown in liquid media expressing and not expressing heterologous ZmEPSPS to validate the desired growth-based selection strategy. Figure 4 This EPSPS-dependent yeast (denoted as sKR-024) is used as the basis for a synthetic yeast model to study known mutants and discover new high-function variants.

[0155] Characterization of the synthetic EPSPS mutation screening system After developing the ZmEPSPS-dependent strain, we then sought to confirm whether this complementation could induce and reproduce glyphosate sensitivity and plant responses in Saccharomyces cerevisiae. To this end, strain sKR-024 was grown at different glyphosate concentrations to determine sensitivity. Compared with WT BY4741, this strain did exhibit a growth defect in glyphosate, but over time, it was ultimately able to show growth even at high glyphosate concentrations ( Figure 1 ). This escape phenotype may be attributed to the high expression of ZmEPSPS achieved here with the strong TEF1 promoter, and is similar to previous literature which observed improved glyphosate resistance in some weeds where wild-type EPSPS had been highly expressed. This observation indicates that this synthetic yeast system can reproduce accurate growth trends from plants.

[0156] To determine whether this system could further confirm the observations from previous literature, well-studied EPSPS mutants T102I and P106S and the combined double mutant TIPS (T102I-P106S) were generated and complemented into this yeast system. These mutations were reported to result in different degrees of glyphosate resistance. As Figure 1 (Figure C) shows, the experimental growth data in yeast was confirmed to generally match the literature trends related to the glyphosate tolerance of these EPSPS variants of P106S < T102I < TIPS.

[0157] Next, we sought to confirm that this tolerance difference led to selectable phenotypes. To this end, a mock selection for glyphosate resistance was achieved by transforming wild-type EPSPS into sKR-024 containing an RFP cassette and TIPS into sKR-024 without the RFP cassette for flow cytometry tracking of population distribution. A 50:50 starting inoculum of each strain was cultured in yeast minimal medium containing 0, 2.5, 5, 7.5, or 10 mM glyphosate. After adding glyphosate in a titratable manner, the count of yeast containing wild-type EPSPS (RFP fluorescence of approximately 104 RFU) began to rapidly decrease ( Figure 5 ). On the other hand, the non-fluorescent strain containing the TIPS variant showed consistent cell counts at multiple glyphosate levels, as predicted by its significantly higher K i .

[0158] The final observation from this initial mock selection experiment was that in the absence of glyphosate, the cell count of TIPS was consistently lower relative to wild-type EPSPS, thus indicating a lower overall fitness for TIPS. Previous literature has reported that mutants leading to higher glyphosate tolerance have lower fitness for K m and / or k catThis has a negative impact. In plant models, the absence of glyphosate results in significant growth defects phenotypically in plants expressing EPSPS mutants. To further explore this phenotype (and to achieve a more robust dual selection strategy), we examined whether lower expression of complementary EPSPS enzymes could further exacerbate this defect and lead to stronger selection pressure. To this end, a series of synthetic promoters were constructed (in order of reported expression levels) consisting of TEF1p, CYC1p, STE5p, and synthetic minimal yeast promoters (Core1p and Core4p). Thus, we found that weaker expression of EPSPS and TIPS was indeed associated with lower specific growth rates, longer lag times, and generally lower final OD values. Figure 6 However, when expression intensity decreases, the growth rate of TIPS decreases much more than that of EPSPS. Figure 6 (Figure A). Therefore, this weaker expression exploits the kinetic defects of TIPS and provides a selection window for discovering EPSPS variants originating from the TIPS background (and other backgrounds). Since the synthetic promoter Core1p produces the largest difference in growth rate between EPSPS and TIPS, it was chosen for all future selection studies.

[0159] Low copy number and weak promoter expression of EPSPS variants have been established, with the aim of recharacterizing the phenotype under field conditions for in situ plant development. Therefore, Figure 6 Figures C and D show the observed growth characteristics of this synthetic yeast model, in which wild-type EPSPS, P106S, and T102I-P106S (TIPS) were expressed in 0.5 mM or 0 mM glyphosate. It has been previously reported that 0.3 mM or more glyphosate (gly) can accumulate in root nodules and meristems of plants under standard glyphosate foliar application. In the yeast basal medium (0 mM gly), the growth of EPSPS and P106S was very similar, supporting the fact that they have similar kinetic properties. However, as observed in the plant host, TIPS growth was significantly defective. On the other hand, in 0.5 mM gly, EPSPS was completely inhibited, showing no growth, while P106S showed slightly higher growth than EPSPS after 80 hours, supporting its 5-fold higher K+ concentration compared to glyphosate. i The values ​​were not specified. TIPS, highly insensitive to glyphosate, grew well in the presence of glyphosate, but still showed overall growth below the WT due to low catalytic efficiency. These trends support the reported literature on the growth of mutants P106S and TIPS in various plant hosts. Therefore, this synthetic yeast model system closely resembles the observations in situ plants and can thus be used to select bitrait EPSPS variants targeting both enzymatic efficiency and glyphosate resistance.

[0160] Flexible selection of EPSP synthases through directed evolution using synthetic yeast systems With host selection in place, the next step was to evaluate various directed evolution strategies to generate novel variants of maize EPSPS. Mutagenic libraries were generated using both EPSPS and TIPS variants as background scaffolds. These mutagenic pools were individually selected to observe independent outcomes toward improved glyphosate resistance and / or improved overall catalytic activity. Figure 7 The study evaluated the mutational and phenotypic outcomes of different selection schemes to confirm that flexible selection criteria (i.e., changing the number of subcultures in non-glyphosate medium relative to glyphosate medium) could potentially bias evolutionary outcomes.

[0161] As a first pass, mutagenic libraries from EPSPS and TIPS backgrounds were grown in increased amounts of glyphosate, starting at 0 mM and ending at 5 mM. Figure 8 and Figure 9 For EPSPS, given that wild-type EPSPS has already conferred high growth in non-glyphosate media, it is necessary to improve tolerance; therefore, the library pools were grown only in the presence of increased levels of glyphosate. In the case of TIPS, oscillation between glyphosate and non-glyphosate conditions was preferred to confer improved growth in non-glyphosate media while maintaining the relatively high glyphosate tolerance already seen in this variant. Multiple parallel pools from these backgrounds were placed in separate flasks according to these selection criteria to observe the mutation outcomes. Figure 10 and Figure 11 ).

[0162] A few novel mutations were found in the EPSPS library pool, including A188T, which is located in the α-helix near P106S and in a position that plays an important role in the conformational changes that can occur when both PEP and S3P bind to the substrates. However, the well-studied proline-serine mutation (P106S) dominated the EPSPS background, thus demonstrating the strength of this synthetic yeast model for selecting glyphosate-resistant mutants. In the TIPS mutagenesis pool, a few novel mutations emerged in this initial first pass, but the selection outcome was primarily TIPS-dominated. Therefore, a better set of exogenous criteria would balance the selection pole, as growth in non-glyphosate environments appears to have lower selection pressure between EPSPS and TIPS than under glyphosate-containing conditions.

[0163] To achieve a better balance, multiple exobiota were investigated in glyphosate-free media, ending with 1-2 exobiota. For this purpose, one culture consisted of a fresh library of TIPS (med) unexposed to any strongly selected medium, one culture consisted of the same library with a single exobiota in 2.5 mM glyphosate, and the last culture consisted of exobiota from… Figure 9 The outcome ended with oscillatory selection at 2.5 mM glyphosate (schematically depicted in...). Figure 12 (Chinese). This experiment aimed to evaluate whether using various starting points led to changes in outcomes and to provide information for better directed evolutionary activities in bitrait evolution. Several interesting observations emerged from the outcomes of these selection strategies (Chinese). Figure 13 , Figure 14 and Figure 15 First, the TIPS mutant was reverted to wild-type EPSPS by selecting four passages at 0 mM and then plated on 0 mM glyphosate agar plates. This demonstrates the utility of this selection pole in improving basal enzyme traits and activities. Second, it was necessary to have a single exogenous organism in liquid medium or agar at high levels of glyphosate to completely eliminate any variants with low glyphosate tolerance, thus validating the strength of this selection pole compared to a non-glyphosate selection pole. More refined titrations at weaker concentrations (such as 0.25 mM) further improved the selection. Third, the resulting mutants were observed to vary according to precise selection pressures and evolutionary trajectories, indicating that each method produced slightly different traits in glyphosate and non-glyphosate media. Identifying a balanced selection pole is important for delivering tailored protein traits, thus necessitating a more sensible strategy for directed evolutionary activities.

[0164] Next, use Figure 16 The optimized selection strategy shown here involves a final directed evolutionary activity starting from an EPSPS or TIPS genetic background using the same library mutagenesis pool. The first stage selects highly functionalized EPSPS variants conferring strong growth traits, and the second stage subcultures the cultures with increased levels of glyphosate to provide hierarchical selection pressure to discover glyphosate-tolerant mutants. The highest hits from each stage of these selections are shown in [the table / data]. Figure 16 Depending on the applied selection pressure, different outcomes were observed. For example, from an EPSPS background, mutants P106S-V125L and P106A-A113V emerged from selection at high glyphosate levels (10 mM glyphosate), while single mutations such as V332A and A188T emerged from selection at lower glyphosate levels (0.5 mM glyphosate). Interestingly, no significant improvement in EPSPS was observed under non-glyphosate conditions, but a few mutants, such as T368A and E225D-Y234F, were isolated after multiple exogenous cultures in non-glyphosate media. It is speculated that selection in non-glyphosate media could reveal variants with better enzymatic efficiency derived from wild-type EPSPS, but these mutations were not observed. For the TIPS background library pool, a wide range of mutation outcomes were observed. Under non-glyphosate selection, TIPS reverted to variants containing only P106S, or completely reverted to wild-type EPSPS, as well as a few other single mutations, such as V377I.

[0165] When the pools were transferred back to a medium containing glyphosate, all resulting variants contained the TIPS mutation plus a few other additional mutations. Notably, the mutants TIPS-V66L-M104L and TIPS-P126S-M217L-K296R were highly dominant in their respective pools and were not observed in the first round of unbalanced oscillatory selection.

[0166] By employing this customized bitrait selection strategy that balances two selection poles, novel mutations were discovered that separated at abundances greater than the starting point EPSPS and TIPS. Interestingly, previously reported single mutations with high glyphosate insensitivity but extremely low catalytic efficiency (such as G101A and T102I) were not observed in any selection pool, further validating the strength of this selection strategy in achieving bitrait phenotypes.

[0167] In vivo characterization of novel mutants from different selection criteria Selected mutants isolated from the final directed evolution activity using an improved selection strategy were further evaluated at various glyphosate concentrations to determine growth characteristics. Mutants isolated from the EPSPS background were evaluated in media containing 0 mM and 0.25 mM glyphosate, the latter being the concentration at which wild-type EPSPS failed to grow after 100 hours. Mutants isolated from the TIPS background were evaluated in media containing 0 mM and 5 mM glyphosate, as the activity aimed to maintain high glyphosate tolerance while enhancing catalytic efficiency. These results are presented as phenotypic profiles. Figure 17 In the diagram, non-glyphosate single selection, highlighted in blue, represents mutations resulting from five subcultures in 0 mM glyphosate. Low-glyphosate double selection, highlighted in light green, represents mutants resulting from additional subcultures in glyphosate up to 0.5 mM. Finally, high-glyphosate double selection, highlighted in dark green, represents mutations that segregated after subcultures in increasing levels of glyphosate up to 10 mM.

[0168] These results confirm that flexible selection criteria can produce customized enzymes. For example, pools grown solely in glyphosate-free yeast basal medium tend to produce phenotypes concentrated in the lower right quadrant of the phenotypic profile, where normal enzyme activity and growth characteristics are dominant. For EPSPS, this resulted in the isolation of wild-type EPSPS as well as several other mutants with similar growth characteristics to EPSPS. For TIPS, reversion of T102I or even complete reversion to EPSPS was observed in selections that were overly biased towards improving catalytic efficiency. Interestingly, single subcultures in 0.5 mM glyphosate, representing a low-glyphosate dual selection, produced novel mutants with moderate glyphosate tolerance that were not dominant in 0 mM glyphosate single selection, such as A118T and V332A from an EPSPS background and TIPS-P126L and TIPS-I177V from a TIPS background. Finally, selection at higher glyphosate concentrations (2.5 mM, 5 mM, and 10 mM) primarily yielded mutants that retained high glyphosate tolerance and also exhibited high growth in non-glyphosate environments, such as TIPS-V66L-M104L and TIPS-P126S-M217L-K296R from the TIPS background.

[0169] In one implementation, the best-performing mutant TIPS-P126S-M217L-K296R and the single mutant TIPS P126S were further rigorously evaluated to determine their specific growth rate compared to EPSPS and TIPS. TIPS P126S / M217L / K296R conferred high glyphosate tolerance (even higher than TIPS) and retained normal growth characteristics in synthetic yeast hosts, similar to EPSPS, with only a slightly prolonged lag time. To identify the potential mutations leading to the highest improvement in growth phenotype here, each combination of the mutant TIPS-P126S-M217L-K296R was then generated ( Figure 18 Furthermore, it was found that M217L did not confer any improved growth traits and may even be slightly detrimental under some conditions. In addition, compared to TIPS, both P126S and K296R, as single mutants, improved growth in non-glyphosate media, and the additive effect of P126S and K296R further improved the growth phenotype to near-wild-type levels. Figure 19 ).

[0170] Next, the mutants P126S and K296R were determined relative to other glyphosate resistance mutant scaffolds (including G101A). Figure 20 ), T102I ( Figure 21 ) and P106S ( Figure 22The portability of the mutations was assessed. These mutations were found to confer improved growth in both 0 mM and 5 mM glyphosate against a background of all known glyphosate-resistant single mutants. Notably, significant improvements were observed in both G101A and T102I at 0 mM, both mutants exhibiting independently extremely low catalytic efficiency and strong growth defects. Furthermore, in some combinations, mutations in P126S and K296R resulted in improved growth in certain variants at 5 mM glyphosate. For example, P106S with the dual mutant P106S-K296R showed growth in 5 mM glyphosate, while P106S itself did not show growth at high levels of glyphosate.

[0171] Finally, the mutant TIPS-P106S-K296R was evaluated at different glyphosate levels, from mild to extreme. TIPS-P106S-K296R performed excellently under every condition with glyphosate levels ranging from 0.05 mM to 5 mM, and, as previously shown, its performance at 0 mM glyphosate was very similar to that of wild-type EPSPS. Figure 23 Therefore, TIPS-P106S-K296R is extremely robust and imparts highly similar growth rates under all conditions, further supporting its potential use in situ in plants.

[0172] TIPS: In vitro characterization and crystallization of P126S and K296R In another embodiment, the highest-performing mutant TIPS-P106S-K296R was characterized in vitro to further understand the improved kinetic and structural basis. As shown in Table 3, the enzymatic efficiency of the mutant TIPS-P106S-K296R was significantly higher than that of the evolutionary origin TIPS, approximately 2.5-fold. The enzymatic activities of EPSPS and TIPS (including the repression constant K) were also compared. i This closely confirms the values ​​previously reported. The mutations P126S and K296R themselves affect K... m and k cat Both have beneficial effects, and these effects are additive when combined, similar to observations of in vivo growth characterization. Furthermore, each variant maintains high K... i The values ​​show similar or slightly higher values ​​of approximately 800 μM compared to the starting point TIPS. These findings confirm the phenotype conferred by the dual-trait selection pressure employed during directed evolution. Kinetic parameters of maize EPSPS with mutations discovered via directed evolution are shown. Table 4 presents triplicate measurements of initial velocity data fitted to the Michaelis equation. The values ​​represent the mean ± SE of the three rate measurements. Regarding K... m The value is obtained by changing one substrate and having another substrate in a saturated state (approximately its K). m To obtain (10 times) and therefore for apparent K m Regarding kcat The values ​​shown are those obtained by substrate saturation with PEP, and they are not significantly different from those obtained with S3P in any case.

[0173] Table 3 To understand how the P126S and K296R mutations improve enzyme activity, all maize EPSPS variants (wild type, TIPS, TIPS-P126S, TIPS-K296R, TIPS-P126S-K296R) were studied using X-ray crystallography. The crystal structure of ZmEPSPS has not yet been reported in the literature; this study is based solely on recent reports from Arabidopsis thaliana. Arabidopsis thaliana The structure of EPSPS31 was inferred through computer simulations based on similarity. Each of the five variants was co-crystallized with shikimic acid-3-phosphate (S3P) and glyphosate to capture states close to the active reaction center. Crystal diffraction was performed, and the final structures of all five variants were refined to a resolution extended to approximately 2 Å (Table 3 above). Due to the protein's low symmetry space group (P1), and the limitations imposed by crystal orientation, only two variants (TIPS-K296R and TIPS-P126S-K296R) could be scaled down to less than 90% integrity (Table 3). However, the density of all ligands in the structures was very clear. Figure 24 This analysis shows that maize EPSPS retains the same conformation as other reported EPSP synthases. As previously observed, the domains are highly dynamic and close upon ligand association. For each mutant, the location and identity of the mutant residues can be clearly distinguished from the high-resolution structure. Figure 25 , Figure 26 and Figure 27 However, the overall folding of the protein is almost identical. The structure of the TIPS variant is also consistent with previous observations that the TIPS mutation confers glyphosate resistance to the enzyme by shifting the position of Gly101.

[0174] Unlike the TIPS mutation site, P126S and K296R are located approximately 14 Å and 26 Å from the active site, respectively, thus making it impossible for them to directly facilitate protein-ligand interactions. Figure 25 However, both mutations stabilize the protein's surface energy and contribute to allosteric enhancement of enzyme activity. For example, P126 is a nonpolar residue located at the sharp β-turn on the surface of EPSPS proteins. The substitution of proline with a small polar residue (such as serine) alleviates the torsional stress of this tight turn, while further stabilizing it through a solvation effect. Figure 25On the other side of the protein surface, K296 is another positively charged residue that can be replaced by arginine. Although the interactions around K296 (or R296) are not definitively defined due to structural flexibility, substitution with arginine may provide better surface stability due to increased electrostatic interactions or hydrogen bonding. Figure 25 ).

[0175] Selection systems are crucial for determining the outcome of directed evolutionary activities. In this embodiment, a synthetic yeast host was developed to characterize and evolve novel EPSP synthase variants. By evaluating the outcomes of multiple selection pressures and multiple mutagenesis pools, this synthetic yeast host enables the selection of EPSPS variants with tunable dual traits via exogenous growth in glyphosate-free or glyphosate-containing media. Using these flexible selections, variants with specific phenotypes can be isolated simply by varying exogenous conditions and the number of subcultures. This results in the isolation of variants ranging from low glyphosate tolerance or low enzymatic efficiency to variants with a combination of high glyphosate tolerance and high enzymatic efficiency. By balancing selection pressures via a dual-trait exogenous phase, well-studied double mutant TIPS evolve to grow better at a wide range of glyphosate exposure levels in a single round of evolution. Thus, a mutational and phenotypic profile characterizes the evolutionary outcome of the protein. For the most promising protein variants, a few mutational changes can surprisingly increase in vitro enzymatic efficiency many times over while maintaining robust glyphosate tolerance. Given these findings, more targeted evolutionary activities (such as targeting the loop region near residues 125-130 that closes upon S3P binding) and additional rounds of mutagenesis may yield productive outcomes with improved phenotypes. This synthetic yeast host can be used for rapid prototyping of novel EPSPS variants that can be further studied in situ in plants. In summary, this example highlights the potential of dynamic selection criteria to tune proteins to specific bitrait phenotypes of interest, and the potential of yeast models as a universal synthetic tool for studying herbicide mechanisms.

[0176] Example 2: Characterization of selected variants in maize plants EPSPS variants were characterized in plant systems to determine whether any of the observed improvements represented in situ biological improvements in plants. Due to the detailed characterization of glyphosate tolerance in maize and suitable transgenic plant production protocols dependent on glyphosate, maize was used as a model system to test the functionality of EPSPS variants. Plant transformation vectors were generated using native maize EPSPS sequences comprising native gene promoter elements, native introns and exons, and a native 3' untranslated region, with mutations corresponding to each of the selected amino acid changes introduced. The native sequences were used to simulate as closely as possible how the selected EPSPS variants would behave if introduced directly into the genome without the aid of strongly expressed elements. The following variants were selected for testing in the transgenic maize system: TIPS (as a control); TIPS-P126S-M217L-K296R; TIPS-P126S-K296R; T102I-P126S-K296R (where only T102I variation exists); P106S-P126S-K296R (where only P106S variation exists); and P126S-K296R (where no TIPS variation exists).

[0177] Transgenic maize plants expressing each of six EPSPS variants were generated to determine whether the variants conferred glyphosate tolerance to the plants. The complete DNA sequence encoding TIPS EPSPS (SEQ ID NO:1) was cloned from maize genomic DNA. In this sequence, the promoter and 5' UTR are nucleotide 1:2556; the chloroplast transport peptide sequence is nucleotide 2557:2742; exon 1 is nucleotide 2743:2856; intron 1 is nucleotide 2857:3384; exon 2 is nucleotide 3385:3626; intron 2 is nucleotide 3627:3725; exon 3 is nucleotide 3726:3879; intron 3 is nucleotide 3880:4152; and exon 4 is nucleotide 41. 53:4367; Intron 4 is nucleotide 4368:4877; Exon 5 is nucleotide 4878:4995; Intron 5 is nucleotide 4996:5155; Exon 6 is nucleotide 5156:5366; Intron 6 is nucleotide 5367:5446; Exon 7 is nucleotide 5447:5508; Intron 7 is nucleotide 5509:5617; Exon 8 is nucleotide 5618:5836; and the 3' UTR is nucleotide 5837:6368. The mutation was then introduced into the EPSPS coding sequence to generate each of the six EPSPS variants to be tested. The whole-genome DNA sequences of these mutations were then cloned as individual expression cassettes into plant transformation vectors.

[0178] Agrobacterium tumefaciens was prepared using methods known in the art. (Agrobacterium tumefaciens)Mediated plant transformation. Glyphosate selection was used to select transgenic items (events) containing the transgene of interest, and only those EPSPS variants that provided effective glyphosate tolerance in plant cells were further screened. Although all six EPSPS variants were started with the same amount of starting maize cell material, the number of recovered plantlets that could be transplanted into greenhouse soil medium differed (Table 4). Transgenic items were further analyzed to determine transgene copy number. Healthy items with a single copy of the transgene of interest were grown in greenhouse soil medium. Only three of the six EPSPS variants had more than one surviving plant. Up to twenty-five of these developing R0 plants were transplanted into larger pots and allowed to mature in a greenhouse for seed collection.

[0179] Two of the six EPSPS variants were used, with more than twenty-five developing plants. Additional developing R0 plants from these two variants were used in a glyphosate spray test to determine the tolerance to typical glyphosate application to young plants provided by the EPSPS variants. At stages V3-V4, R0 plants were sprayed with post-emergence (POST) glyphosate at 0.75 lb ae (acid equivalent) / ac (0.84 kg ae / ha). Damage to the treated plants was evaluated 7 days after glyphosate application.

[0180] The results showed that the two experimental EPSPS variants, TIPS-P126S-M217L-K296R and TIPS-P126S-K296R, were able to provide glyphosate tolerance, enabling healthy plants to successfully transform and grow to produce seeds. One of these experimental EPSPS variants, TIPS-P126S-K296R, produced seedlings showing better glyphosate tolerance than the TIPS control, with 0% of the sprayed plants showing damage compared to 30% of the TIPS control plants (Table 4). Results are shown in... Figure 28 middle.

[0181] Table 4 Example 3: In-situ plant testing of selected EPSPS variants in maize To further evaluate the efficacy of EPSPS variants in glyphosate tolerance, transgenic maize plants containing a single copy of the transgene encoding either EPSPS variant TIPS-P126S-M217L-K296R or TIPS-P126S-K296R were grown to maturity. Homozygous transgenic plants were then crossed with inbred lines to produce hybrid seeds.

[0182] Plants from hybrid seeds (hemizygous for transgenics) of two independent transformations from each of the two variants were grown in a greenhouse for glyphosate spray testing. Roundup® PowerMax3, a commercial formulation of glyphosate, was applied at the third leaf stage at two rates: 1120 g / ha (1x field rate) and 2240 g / ha (2x field rate). Controls included an untreated, unconverted control without glyphosate treatment, an unconverted control sprayed with glyphosate, and a TIPS positive control sprayed with glyphosate. Glyphosate herbicide damage (stunting, yellowing, necrosis, deformity, and death) was assessed by visual inspection of plants 18 days after herbicide treatment. Herbicide damage rates were presented on a scale of 0 to 100, where “0” represents no visible crop damage and “100” represents complete crop damage or death.

[0183] The results of the glyphosate spray test are summarized in Table 5. Both the TIPS-P126S-M217L-K296R and TIPS-P126S-K296R variants provided excellent tolerance to the equivalent 1x field dose of glyphosate. Furthermore, the TIPS-P126S-M217L-K296R variant provided complete tolerance to 2x field doses, indicating robust tolerance. Both TIPS-P126S-K296R and the TIPS control plants showed slight damage at 2x field doses. This test confirms that both the TIPS-P126S-M217L-K296R and TIPS-P126S-K296R EPSPS variants provide robust tolerance in greenhouse tests designed to simulate field conditions.

[0184] Table 5 - Efficacy of EPSPS variants after glyphosate application The embodiments described illustrate certain implementations of this disclosure. Those skilled in the art will understand that many modifications can be made to the specific embodiments disclosed while still obtaining similar results. Certain chemically and physiologically relevant agents can be used to replace the agents described herein, while achieving the same or similar results. All such substitutions and modifications that are obvious to those skilled in the art are considered to be within the scope of this invention.

Claims

1. A recombinant DNA molecule encoding a glyphosate-tolerant 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS), wherein the EPSPS comprises at least a first combination of amino acid substitutions selected from the group consisting of T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-P126S-M217L-K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the positions of the amino acid substitutions are relative to the positions of the amino acid sequence provided as SEQ ID NO:

6.

2. The recombinant DNA molecule of claim 1, wherein the EPSPS is a corn EPSPS.

3. The recombinant DNA molecule of claim 1, wherein the EPSPS confers increased glyphosate tolerance or increased enzymatic efficiency compared to an EPSPS lacking the combination.

4. The recombinant DNA molecule of claim 1, wherein the EPSPS comprises at least two of the combinations of substitutions.

5. A glyphosate-tolerant EPSPS comprising at least a first combination of amino acid substitutions selected from the group consisting of T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-P126S-M217L-K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the positions of the amino acid substitutions are relative to the positions of the amino acid sequence provided as SEQ ID NO:

6.

6. The glyphosate-tolerant EPSPS of claim 5, wherein the EPSPS confers increased glyphosate tolerance or increased enzymatic efficiency compared to an EPSPS lacking the combination or compared to an EPSPS containing only T102I-P106S.

7. The glyphosate-tolerant EPSPS of claim 5, wherein the EPSPS comprises at least two of the combinations of substitutions.

8. A plant, seed, cell, plant part, or commodity product comprising the recombinant DNA molecule of claim 1.

9. The plant of claim 8, wherein the plant exhibits increased glyphosate tolerance when compared to a plant lacking the combination.

10. The plant of claim 9, wherein the plant is a maize, soybean, cotton, canola, wheat, rice, alfalfa, sugar beet, rapeseed, or sugarcane plant.

11. A transgenic maize plant comprising a recombinant DNA molecule encoding a glyphosate-tolerant 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS), wherein the EPSPS comprises at least a first combination of amino acid substitutions selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-P126S-M217L-K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the positions of the amino acid substitutions are relative to the amino acid sequence provided as SEQ ID NO: 6, and wherein the EPSPS confers glyphosate tolerance to the transgenic maize plant.

12. The transgenic maize plant of claim 11, wherein the glyphosate tolerance is greater than that conferred by the EPSPS comprising only the T102I-P106S substitution.

13. A seed, cell, or plant part of the plant of claim 11, wherein the seed, cell, or plant part comprises the recombinant DNA molecule.

14. A method for conferring glyphosate tolerance to a plant, the method comprising expressing in the plant the glyphosate-tolerant EPSPS of claim 5.

15. The method of claim 14, comprising introducing into the genome of the plant or its ancestor the recombinant DNA molecule of claim 1.

16. A method for producing a glyphosate-tolerant EPSPS, the method comprising introducing into a nucleic acid molecule encoding a plant EPSPS at least a first amino acid substitution combination selected from the group consisting of: T102I-P106S-P126S-K296R, T102I-P106S-P126S, T102I-P106S-K296R, T102I-P106S-P126S-M217L-K296R, T102I-P106S-V66L-M104L, T102I-P106S-T108S, T102I-P106S-F150V-H318Y, T102I-P106S-P126L, T102I-P106S-G116V, T102I-P106S-I177V, and P106S-A109V-I163K, wherein the positions of the amino acid substitutions are relative to the position of the amino acid sequence provided as SEQ ID NO:

6.

17. The method of claim 16, wherein the introducing is performed in vitro.

18. The method of claim 16, wherein the introducing is performed in planta.

19. The method of claim 16, wherein the introducing comprises using at least a first site-specific endonuclease.

20. The method of claim 16, wherein the method comprises introducing at least two of the substitution combinations into the EPSPS.

21. A method for controlling weeds in a plant growing area, the method comprising contacting a plant growing area comprising a plant or seed of claim 8 with glyphosate, wherein the plant or seed is tolerant to glyphosate, and wherein weeds are controlled in the plant growing area.

22. A yeast cell comprising a knockout of native arol function and further comprising a recombinant DNA molecule comprising a heterologous promoter operably linked to an arol coding sequence comprising a D731A mutation, wherein the yeast cell is unable to grow on media lacking aromatic amino acids.

23. The yeast cell of claim 22, further comprising a heterologous nucleic acid molecule encoding an EPSPS, wherein the yeast cell is capable of growing on media lacking aromatic amino acids.

24. The yeast cell of claim 22, wherein the EPSPS is a glyphosate-tolerant maize EPSPS.

25. The yeast cell of claim 22, wherein the yeast cell is a Saccharomyces cerevisiae cell.

26. A method for identifying a glyphosate-tolerant EPSPS having improved growth, improved glyphosate tolerance, or improved enzymatic efficiency when compared to a wild-type EPSPS, the method comprising the steps of: a) obtaining a yeast cell of claim 22; and b) contacting the yeast cell with glyphosate. b) identifying the yeast cell as being capable of growing in the presence of glyphosate and thus comprising a glyphosate-tolerant EPSPS having improved growth, improved glyphosate tolerance, or improved enzymatic efficiency when compared to wild-type EPSPS.

27. The method of claim 26, wherein the method comprises obtaining a population of yeast cells collectively comprising a plurality of heterologous nucleic acid molecules encoding a mutant EPSPS protein, and identifying at least one of the yeast cells as having improved growth, improved glyphosate tolerance, or improved enzymatic efficiency.

28. The method of claim 26, further comprising cloning a nucleic acid molecule encoding the glyphosate-tolerant EPSPS from the yeast cell or a progeny thereof.

29. A method of identifying a plant, seed, cell, or plant part as comprising glyphosate tolerance, the method comprising applying glyphosate to a plant, seed, cell, or plant part comprising the recombinant DNA molecule of claim 1.

30. The method of claim 29, further comprising applying the glyphosate to a population of plants, seeds, cells, or plant parts.

31. The method of claim 30, comprising applying the glyphosate to a cell culture.

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